The Taxonomic Status of Gladiolus Illyricus (Iridaceae) in Britain

The Taxonomic Status of Gladiolus illyricus (Iridaceae) in Britain

Aeron Buchanan

Supervisor: Fred Rumsey, Natural History Museum, London A thesis submitted in partial fulﬁlment of the requirements for the degree of Master of Science of Imperial College, London Abstract

First noticed ofﬁcially in Britain in 1855, Gladiolus illyricus (Koch) presents an interesting taxonomic and biogeographical challenge: whether or not this isolated northern population should be recognized as a separate sub-species. Fundamental conservation issues rest on the outcome. Here, the investigation into the relationship of the G. illyricus plants of the New Forest, Hampshire, to Gladiolus species across Europe, northern Africa and the middle east is initiated. Two chloroplast regions, one in trnL–trnF and the other across psbA–trnH have been sequenced for 42 specimens of G. illyricus, G. communis, G. italicus, G. atroviolaceus, G. triphyllos and G. anatolicus. Phylogenetic and biogeographical treatments support the notion of an east–west genetic gradation along the Mediterranean. Iberia particularly appears as a zone of high hybridization potential and the source of the New Forest population. Alignment with sequences obtained from GenBank give strong support to the classic taxonomy of Gladiolus being monophyletic in its sub-family, Ixioideae. Comments on these chloroplast regions for barcoding are also given. In conclusion, the genetic localization of Britain’s G. illyricus population as an extremity haplotype suggests that it could well deserve sub-species status.

Contents

1 Introduction 2

2 Background 4

3 Materials and Methods 8

4 Results and Discussion 15

5 Conclusions 26

Appendices 28

References 56 1. Introduction G. illyricus in Britain

Figure 1: G. illyricus ﬂower spike. Photograph: Fred Rumsey, Denny Wood, New Forest.

1 Introduction

The study of Gladiolus illyricus (Koch) is particularly pertinent at this time. It is a medium-sized flowering monocotyledon with a Mediterranean distribution, but with one important exception: the small yet significant, and now declining population in the New Forest of Hampshire, here in the British Isles. The natural history of G. illyricus in the UK, until as recently as the 1950s, ranges from being unclear to unknown. The first recorded sighting was on the Isle of Wight in 1855, with notes of its presence in the New Forest appearing soon thereafter. The origins of this population, in the extreme north of the species’ range, immediately became a point of debate. Was the arrival part of a natural progression northwards or was it an artificial, human-mediated introduction? Today, the continuing survival of G. illyricus in the New Forest appears fragile, making the above an important question. It is currently a protected Schedule 8 species and so protected under law by the Wildlife and Countryside Act, 1981, but with its endemic status unresolved, it remains on the waiting list of Cheffings and Farrel (2005)∗. The waiting list is populated by “taxa for which questions still remain over taxonomic validity or endemic status.” Indeed, the example of such a taxon given by the authors is G. illyricus ssp britannicus. This state of conservation limbo is unsatisfactory, for if the endemic sta-

∗‘The Vascular Plant Red Data List for Great Britain’ is part of the Joint Nature Conservation Com- mittee’s Species Status project, which is used to inform the statutory ﬁve-yearly review of Schedules 8 species.

2 1. Introduction G. illyricus in Britain tus is rejected then much demanded conservation resources can be redirected elsewhere. However, if it is conﬁrmed, a fuller more concerted effort would be justiﬁed. The issue of the British nativity of G. illyricus is theoretically inseparable from the suggestion that the population in the New Forest should be designated as being a separate subspecies, G. illyricus ssp britannicus. This study was born with the desire to resolve the taxonomic status of G. illyricus and hence shed light on the natural history of the New Forest population. Resolution would thus make clear the level of impetus required of conservation efforts. An outline for a study to answer the question has been proposed by Lockton (2006); that is, to demonstrate:

1. the existence of an east–west genetic gradation 2. that the gradation is correlated to natural dispersal rates. 3. that there is speciation within that gradation. 4. that the British plants are sufﬁciently distinct. 5. that they belong to a distinctive semi-natural vegetation habitat.

The ecological investigation, that can make clear the issues of the fifth point has already started, most notably by Stokes (1987). This study begins the molecular investigation of the G. illyricus population of the New Forest, and hence starts to address the issues of the first, third and fourth points. The work began with the important grounding step of placing the European Gladiolus species into a wider context. Gladiolus is one of the largest genera in the Iridaceae family, with possibly as many as 300 species. Almost all of these grow in sub-Saharan Africa (Goldblatt et al. 2001); less than twenty species are endemic to Europe and neighbouring countries. Support for the monophyletic status of all Gladiolus is needed to be able to rely on existing work that assumes the long standing taxonomy. After the broad initial analysis, the project then focussed on the inter-relations within Europe, which were then explored with the above framework in mind. Sadly, the full answer can not be given here: limited time restricted the scope of what was achievable. However, important insights into the wider situation are revealed, providing some answers and raising significant new ones, thus informing future research.

3 2. Background G. illyricus in Britain

Figure 2: An example of G. illyricus from the herbarium at Reading University. This is a New Forest plant, collected in 1960.

2 Background

The first systematic study of Gladiolus illyricus (Koch) growing in the UK did not start until almost one hundred years after it was first officially noticed. As such, our knowledge of its distribution and ecology is very sparse for times before 1950, when Bowman started the surveying of the New Forest for this purpose (Rand, 2005), and almost completely blank for anything before 1855, when Mrs Phillipps of the Isle of Wight made the first record of its growing in the British Isles (More, 1862). According to Townsend (1904) the first mainland discovery was in 1856 by Rev. W. H. Lucas. From the mid to the end of the 19th century, the story seems to be one of undirected specimen collection with the occasional serendipitous discovery of new sites. The Natu- ral History Museum’s British Herbarium holds thirty nine specimens from this period (the first being the 1858 sheets of J. T. Boswell-Syme), all collected from within the New For- est (v.c.11), seldom with a more detailed description of location than “near Lyndhurst”. Accompanying notes are generally sparse as well, although Rand (2005) points out that descriptions of habitat, beyond “amongst bracken”, were sometimes made. The most detailed of these are by Dyer and Trimen (1864), but they worked on only two sites in the New Forest, within less than 3km of each other. Later, Townsend (1904) compiled a summary of the New Forest locations where it had been found, describing three almost distinct areas of modest size, all within 6km of Lyndhurst. Crucially, Pope et al. (2005)

4 2. Background G. illyricus in Britain notes that it was also found near Ensbury, Dorset (v.c.9) around 1874 and collected trice more from the Isle of Wight in 1872, 1897 and 1931, although the last collection was rather haphazard. The plants seems to have then received little attention from the turn of the century until the systematic surveys of Bowman from the 1950s onwards, Hamilton in the 1960s and Everett in the 1980s (Rand, 2005), as well as Stokes (1987). From their foundational reports and the continued efforts of the New Forest Study Group, we are now fairly certain of the full extent of the sub-populations growing in the New Forest. The widespread activity of botanists across Britain means that we can now be sure that G. illyricus no longer grows outside this area. The current range, while still small, is several times larger than that investigated by botanists at the turn of the century, spreading up to 12km from Lyndhurst. What the actual distribution was in those early years is almost impossible to deduce, but it seems reasonable to surmise, with sub-populations growing outside the New Forest at the time, that it was more consistently widespread then, than it is today. However, this is a pivotal historic factor for the question of G. illyricus being endemic in the New Forest: care must be taken before assumptions are made. Was the sudden start of botanical interest in the middle of the 1800s perhaps a result of a contemporary introduction to the south coast from a continental population? I believe not: it is most likely due to artifacts of historical circumstances and not because of any events or trends belonging to the plants themselves over the period in question. Indeed, Mrs Phillipps did not luckily stumble upon the singular occurrence of G. illyricus on the Isle of Wight, but rather that the “Wild Gladiolus” plant had been growing in the woods at Shanklin, the nearby town, for at least a generation (More, 1862). It is quite possible that, had local people written of the flowers that they commonly saw, or such notes more widely disseminated, records of many plants would have appeared much earlier. Martin Rand (2005) makes a good case for the existence of G. illyricus in the New Forest before the first records appeared. He points out that many species, clearly native in the New Forest, were not officially noticed as growing there for a very long time (Carex montana: 1876 and Gallium constrictum: 1924, for example). He adds to that a description of the difficulty of finding G. illyricus colonies, even when in flower. Another possibly relevant issue is that access to the New Forest became easier after the railway through it opened in 1847. Furthermore, Rand collects a variety of ecological observations to support the idea that G. illyricus has been in Britain for some time. Despite its restricted distribution, it appears in a range of ecotones with a variety of associations, none of which are guaranteed. Also, it seems to bare an interesting relationship with semi-natural habitats (some dating back thousands of years), to which it

5 2. Background G. illyricus in Britain is seemingly well adapted. Overall, a strong sense of long-term naturalization is given by what we do know, although the evidence frustratingly does little to dismiss the neophyte hypothesis outright. However, the notion of a very recent introduction can be disregarded: that there was a prolific dispersal of a plant sensitive enough to just about immediately fall into decline, or that there was some artificial, yet persistent effort to spread the flower across Hampshire are highly improbable. It seems certain, therefore, that it did not arrive just in time for the botanists getting off the first trains ever to arrive at Lyndhurst, but has it been here long enough to be considered native? The debate on the endemism of G. illyricus started alongside the first reports of its discovery. Alexander More published his opinion in the article revealing the 1855 specimen (More, 1862). He was convinced that it was indigenous to Britain: the remoteness of the Isle of Wight location, the lack of any known garden cultivations and the similarity of the growing environment between the plants in the UK with those undisputable natural populations in north western France, together strongly supported the notion of a natural progression along the Atlantic coast from the Mediterranean. As Toone notes in his review of this topic (2005), by the end of 1863 Borrer, Babington and Boswell-Syme had all agreed that the case for it being a native species was strong. Mansell-Pleydell (1874) added him- self to this list in his ‘Flora of Dorset’. H. C. Watson provided the only opposition, suggesting the possibility of an association with planted trees or shrubs. Townsend (1904) reported the idea that it could have been introduced, along with other species, with the imports, around 1800, of young fir trees to Bournemouth from the Landes area of France (where a similar G. illyricus was known to grow). However, evidence does not support any correlation between the growing areas of these two species. As such, I think it is reasonable to assume that the first records were not an account of a spreading introduction, but rather the initial documentation of an established population. A picture emerges that suggests a lower bound on the age of the Britain plants is of the order of many hundreds of years. It could be considered even higher, depending on the rating of the likelihood of jump dispersal events. Assuming that the Isle of Wight population was once a part of a mainland range puts the lower bound at being over 7000 years ago (Cooper and Jay, 2002). Rejecting long range dispersal altogether and therefore assuming that G. illyricus arrived in Britain by migration, fixes the arrival time to before the last formation of the Channel, about 8000 years ago. The upper bound is a little more certain, being defined by the timetable for the increase in temperatures since the last glacial maximum, over which the ice sheet and permafrost retreated north allowing plants growing in the refugia along the Mediterranean to rapidly

6 2. Background G. illyricus in Britain spread north and recolonize Europe (Hewitt, 1999). Temperatures started to rise again from about at least 18,000 years ago (the lower bound for the last glacial maximum or LGM), at which point southern Britain, connected to France at the time, was within the limits of the permafrost and mostly covered by the ice sheet of northern Europe (Taber- let et al. 1998). After a period of warming, the Younger Dryas stadial (Berger, 1990) meant Britain still had sub-zero mean annual temperatures until about 11,500 years ago (Atkinson et al. 1987). This ended abruptly when temperatures rose by as much as 7◦C in a matter of decades and then steadily increased to near present averages (Alley, 2000). The climate of the south coast of England permanently reached temperatures suitable for G. illyricus maybe as soon as 1000 years later. Migration is an additional limiting factor: after the LGM, the maximum migration rates achieved were an impressive, but bounding, 2km per year (Bennett et al. 1986). It seems, therefore, that G. illyricus was probably not growing in the precursor of the British Isles much before 10,000 before present. How long after this G. illyricus could have actually arrived would be nothing more than guess at this point. Today, there is a deﬁnite trend of decline (Brewis et al. 1996). For example, a search in 1947 concluded that it gone extinct on the Isle of Wight since the last sighting in 1931. Within the New Forest itself, the smaller sub-populations are slowly disappearing. The matter might be all the more pressing for the fact that its decline is also being seen in France. Sub-populations are known to have gone extinct, including the crucial Belle Isle (Brittany) population, which is thought to be the most closely related continental population to the British plants (Stokes, 1999). Like across the whole of North West Europe, a huge percentage of France’s heathland has been lost over the last 200 years (mainly to agriculture) and this is undoubtedly a signiﬁcant factor. A trip to France undertaken to collect samples for this project at known, albeit almost historic, locations (Abbayes et al.; Boreau, 1857; Coste, 1906; Lloyd, 1844; Lloyd, 1886; Souche,´ 1901) found that in most places, areas of suitable habitat were nowhere to be seen. The most promising locations were on rocky, heathered slopes above the River Argenton between Argenton-Chateauˆ and Thouars, but no sign of any Gladiolus were found. This author would not be sur- prised to discover that G. illyricus is extinct in the Loire Valley. Hence the importance of determining the resources and effort that should be spent on the conservation of the species here in Britain now.

7 3. Materials and Methods G. illyricus in Britain

number of specimens 12 successfully sequenced (total = 49) failed to sequence (total = 17) 5

0 year 2007 2003 2000 1999 2004 1996 1995 1993 1992 1990 1989 1986 1985 1984 1982 1981 1980 1979 1978 1976 1974 1973 1972 1970 1967 1960 1958 1950 2005 2001 1997 1994 Figure 3: Success in the sequencing of the herbarium specimens, inﬂuencing which were selected for processing. Keeping track of sequencing successes meant that it could be safely guessed that the last six oldest samples were unlikely to succeed and effort was spared by not attempting extractions. 3 Materials and Methods

Leaf samples were obtained from two sources. Firstly, a licensed field trip to the New For- est at the end of the fourth week in June provided 52 small leaf samples, each stored separately and immediately in silica filled bags. All New Forest specimens were identified as G. illyricus. Locations are listed in Appendix A. Secondly, samples of G. illyricus, G. communis, G. italicus, G. atroviolaceus, G. triphyllos, G. anatolicus, G. imbricatus and G. palustris, collected from around the Medi- terranean over the last fifty years, were taken from specimen sheets in the herbarium at the University of Reading. Full details are listed in Appendix B. Selection was mainly dictated by availability. Specimens collected more than fifty years ago were omitted due to the poor outlook on DNA sequencing success, although specimens that might potentially have become key to uncovering relationships were kept. Sheets that were overtly of poor quality or paucity were also overlooked. Sixty two sheets remained from which 0.5–3cm2 of leaf material was taken. Care was also taken to ensure that the original determination was plausible. Six specimens were considered incorrect and eleven were possibly incorrect (notes are given alongside the relevant entries in the appendices). Tutin et al. (1980) was mostly used for the keys, with the complication that only a subset of the characters could be used, because the

8 3. Materials and Methods G. illyricus in Britain plants were dried herbarium specimens. Many of the taxonomically informative distinc- tions between these species are in the morphology of the ﬂowers, including the perianth. Those used for differentiation were as follows. Between G. illyricus and G. communis:

plant height leaf length leaf width no. ﬂowers G. illyricus 25–50cm 10–40cm 4–10mm 3–10 G. communis 50–100cm 30–70cm 5–22mm 10–20 i.e. whether the specimen was large or small. The distinction between G. communis ssp. communis and ssp. byzantinus was not made, because over the limited range and quality of specimens available, a consistent separation was not observed. The principal difference used for the differentiation of G. italicus from G. communis (both having a highly overlapping range of values for the above size characters) was the anther-ﬁlament length ratio:

G. communis anther shorter than ﬁlament G. italicus anther longer than ﬁlament

Ambiguity arose when they were the same length, although exact measurements were not taken because the of the fragility of the herbarium specimens and the invasiveness of such measuring. The one G. palustris specimen was very hard to distinguish from G. illyricus. Differences between the other species encountered were more deﬁnitive. Key characters used include: species most distinct character notes

G. atroviolaceus flowers deep violet-purple well preserved on herb. sheets G. anatolicus seeds unwinged from note on herbarium sheet G. imbricatus dense flower spike but few flowers G. triphyllos leaf width<5mm noticeably “straggly”

The last species, G. triphyllos, is endemic to Cyprus and so was keyed out using Meikle (1985).

Primer Selection and Sequencing

To ensure success of the field collecting, the New Forest trip had to wait until it was certain that any potentially flowering plants were actually in flower. The knock-on effect was to delay the laboratory work. As such, reliable markers were chosen for sequencing targets. Reliable in this context means high copy, monomorphic genome regions of which

9 3. Materials and Methods G. illyricus in Britain the laboratory had experience (there would have been no time for experimentation and optimizations). Three chloroplast regions were picked out as ideal targets: • trnL–trnF (Taberlet et al. 1991) 0 0 (forward) “C”: 5 3 0 0 (reverse) “F” : 5 3 • trnC–trnD (Demesure et al. 1995; Shaw et al. 2005) 0 0 (forward) ‘trnC’: 5 3 0 0 (reverse) ‘ycf6’: 5 3 • psbA–trnH (Sang et al. 1997; Shaw and Small, 2004) 0 0 (forward) ‘psbA’: 5 3 0 0 (reverse) ‘trnH’: 5 3 These are all universal plastid primers. The trnLF region encompass the trnL in- tron, the 30 trnL exon and the trnL–trnF intergenic spacer. For the Gladiolus species sequenced it was found to be 733–742 bases long. It is a popular region, but very unpre- dictable in its variability (Shaw et al. 2005), showing very low rates of change for some groups (e.g. Sang et al. 1997). In the trnCD region, the trnC–ycf6 non-coding region was selected as being likely to be of a good size for trouble-free sequencing (i.e. 500– 1000 base pairs long). This is a less used region and cross-referencing its use is made tricky by the fact that the ‘ycf6’ gene is also known as ‘petN.’ Lee and Wen (2003) have a reverse primer for the same region that they call petN2R which overlaps greatly with the one used here, but whose ends are shifted distally about six bases. The primers employed for this study isolate the trnC–ycf6 intergenic spacer and some of the ‘ycf6’ gene itself. Finally, the psbAtrnH region has been highlighted as a good region for barcoding angiosperms (Kress et al. 2005). It is considered the most variable region of the three plastid regions above. From the results of this study, northern hemisphere Gladiolus have a slightly higher than average number of bases in this region: 609–616. When results from the chloroplast primers were ensured, an ITS region was also investigated. Although, almost always polymorphic (chromosome heterogeneity and di- verging paralogues), it often shows high intra species variation, making it an excellent marker for tracing relationships (Baldwin et al. 1995). Furthermore, because it is a nuclear gene region, information from would perfectly compliment that from the chloroplast sequences, because of the differences in inheritance. • ITS (Douzery et al. 1999) 0 0 (forward) ‘AB101’: 5 3 0 0 (reverse) ‘AB102’: 5 3 Total DNA extraction was performed on all but the oldest of the herbarium specimens

10 3. Materials and Methods G. illyricus in Britain and at least one leaf sample from every location visited in the New Forest, as noted in their respective appendix tables. Extractions proceeded according to a modified CTAB protocol, described in Appendix D. The regions of interest were amplified in polymerase chain reactions (PCRs), the pro- cedures and programmes for which are given in Appendix E. Checks of success of the PCR reactions was carried out by inspecting the presence of PCR products using 0.7% agarose gel electrophoresis. The primer pairs for trnLF and psbAtrnH gave consistently good PCR products. Products from reactions with trnCD were very erratic and this marker was dropped. Its relate position on the gels shared with trnLF and psbAtrnH suggest that it was roughly a little over a 1000 base pairs long for the sequences for which PCR was successful. Attempts at sequencing failed. Attempts to obtain PCR products with the ITS completely failed with all herbarium specimens tried (those with the strongest results using the chloroplast markers were chosen). Results were obtained with the New Forest samples. As expected, several bands appeared on the gel: four distinct bands fading into what could have been several others. The estimated lengths of those that could be identified varied from 1100 down to 500 base pairs. The brightest band (which was also the longest copy) for each specimen was removed and cleaned used the protocol described for the cleaning of extracted total DNA, i.e. band extraction. The gel extracted DNA was of very low quality and did not give any useable sequences. It is suspected that these primers are too widely spaced and that the ITS internal primers (White et al. 1990) should be used. Clean up of the chloroplast PCR products was carried out by the Sequencing Lab- oratory of the Natural History Museum. Once returned, the clean PCR products were re-amplified, but with only one primer in each reaction to obtain quantities of each strand of the DNA separately. These secondary PCR “sequence reactions” are described in Ap- pendix F. The resulting single-strand amplification mixtures were then returned to the Sequencing Laboratory, where they were cleaned again and sequenced. The sequences obtained for this study were thus based on both the forward and reverse strands to maximize fidelity. Some specimens were sequenced more than once to verify work practices. The raw sequence data was checked and edited manually using DNAStar’s ‘SeqMan Pro’ from their ‘Lasergene’ suite. All successful PCR products gave good sequence trace data, although it is important to note that near the start of trnLF an A homopolymer sometimes reached ten bases long, causing slippage during replication. The combination of double-stranded sequences and accounting for the proximal single-base shift algorith- mically meant that this problem was easily overcome when encountered.

11 3. Materials and Methods G. illyricus in Britain

Alignments and Analysis

Alignments of the sequences obtained for the trnLF and trnCD regions were obtained using the server at http://align.genome.jp/ to run CLUSTALW (Thompson et al. 1994) with the ‘slow and accurate’ option and default penalty settings (the differences between the sequences are so small that the alignment is almost insensitive to the adjustment of the penalty settings). All alignments are given in Appendix G. Unique haplotypes were identified using a custom computer script (Appendix H). This program also calculated the change counts between all pairs of sequences. The haplotype network describing all possible intermediate haplotypes was constructed by hand using the output of the program. In the creation of the network, insertion/deletion events (indels) were counted as one change. Furthermore, RH23 displayed evidence of a single 51 base reverse complement event (producing eight aligned nucleotide differences), noticeable from the reverse compliment ‘palindromic’ nature of the region indicative of stem-loop hairpin secondary structure-mediated illegitimate plastid recombination (Klechner, 2000), which was, again, counted as single change. GenBank was searched for additional sequences in an attempt to maximize the achievable resolution in the analyses. Sadly, there were very few Gladiolus DNA sequences on GenBank and only one sequence matching either of the two plastid regions used here. This was a trnLF sequence, and along with the same region for representative species of all the genera in G. illyricus’s sub-family (Ixioideae) and its sister sub-family (Nive- nioideae), were downloaded. See Appendix C and the results section for a complete list. These sequences were aligned by hand, along with all the trnLF haplotypes discovered for this project, in the ‘MacClade’ (Maddison and Maddison) software program. After alignment, the data matrix was purged of unreliable and unusable positions. This was undertaken with the class of analysis that was going to be undertaken (for the sub- family level analysis) in mind: basic cladistic analysis of an unweighted, fixed character data matrix. All that was being sought for this project was direct evidence for the place of Gladiolus species in the wider taxonomic picture, adding a small amount of genetic data to the wealth of morphological evidence that already exists. More sophisticated and in-depth analysis tools, such as simultaneous alignment and relationship derivation (e.g. Wheeler, 2003) or probabilistic methods (e.g. Huelsenbeck, 2001) were considered excessive for the modest task at hand. The notorious issues of weighting, particularly of weighting indels over substitutions, was entirely sidestepped for want of simplicity and straightforward analysis. This is almost justified by taking the process in this case to be one of abstracted evolutionary event counting, rather than detailed evolutionary history

12 3. Materials and Methods G. illyricus in Britain reconstructions. Some of the sequences taken from GenBank were of very poor quality towards their ends. Therefore, the first characters to be excluded from the family-wide trnLF data matrix were the first fifty and last eighty characters. Next, all regions where satisfactory alignments were not possible, namely where ambiguity meant arbitrary assumptions of relationships were required a priori to pick out one alternative over another (which would explicitly invalidate any cladistic analysis) were removed. For example the trnLF region has, in these species, near the beginning, a homopolymer of As of critical length (ten), making it a dubious site for homology signal (Klechner, 2000). Instability is accumulated as DNA polymerase works over such regions, leading to slippage and therefore greatly increasing homoplasy potential well above normal thresholds. The situation was com- plicated by the number of Gs immediately following it varying as well. As such, this homopolymeric region was also excluded. There is also a short dimer repeat, which was retained as it was deemed to be too short (three to six repeats long) for secondary structure complications to have occurred (evidence of any complications was not seen in any of the raw sequence traces). It was coded as an ordered multistate character counting the number of repeats, in this case of AT, so as to avoid the double-weighting that would have otherwise ensued. Indel segments were coded as single binary characters to avoid their excessive and inconsistent overweighting (replacing the problem with the lesser one of possible underweighting). Single base indels, where more than one base class was observed when one was present, were excluded because of the incompatibility of indel and substitution events. Given that characters were not going to be weighted, it is dubious as to whether this is actually an issue in this case, but logically the distinction is an important one. Individual positions where just a single taxon had a gap were included however, because the incompatibility issue was irrelevant due to the gap transition being an autapomorphy and therefore not influential on tree topology. Large multiple position indels with high, but not complete similarity were coded as a single binary character. The information lost almost exclusively differentiated the outgroup taxa only and so was of low relevance. Of the 1272 characters resulting from the alignment, 1192 were excluded and/or parsimony uninformative, leaving 80 characters for optimizations. The number of taxa included was 33: many of the New Forest and Reading Herbarium specimens shared the same character states for these 80 characters and so together they provided just five unique taxa for the data matrix. Including character-set repeats slows down tree searches and in- flates retention indices.

13 3. Materials and Methods G. illyricus in Britain

Cladistic analysis was performed using the computer program PAUP* 4.0b (Swof- ford, 2003). Heuristic searches were performed using Tree Bisection Reconnection (TBR) branch swapping and random stepwise addition sequences. Fitch optimization was employed, with accelerated transition (ACCTRAN) breaking ambiguous optimizations. A total of over 10,000 replicates were run, finding one main island holding 20 trees of length 183. A string of about 68 small islands (holding one or two most parsimonious trees) were also found, none of which changed the strict consensus generated by the main island set. The main island was found on more than 90% of replicate searches. This hit rate of above 9 in 10 suggested that the order of 10 replicates would effectively guarantee its discovery, thus informing replicate number choice for tree topology support analyses. Bremmer support was calculated using ‘TreeRot’ (Sorenson, 1999). Bootstrap analysis proceeded using 200 replicates, each with 100 random starts. The low number of replicates was forced by the very long compute times for some of the bootstrap runs (measured in days). I am not content with bootstrap analysis. It is used as an investigation of a tree topology’s sensitivity to perturbations of character data. If a topology is highly dependent on very few characters then it is likely that low bootstrap support will be attributed to the groupings in question. However, the bootstrap process is only valid when we have all the data and hence know the answer anyway. As such, bootstrap output can be misleading, for example giving low support to good clades (Kitching, 1998). Bootstrap’s overweighting of random characters does not, in my view, reflect anything attributable to real world processes under scrutiny. Jackknife analysis, on the other hand, while suffering from some similar flaws, does at least address this last point. By ignoring some characters completely, jackknifing almost tests the possibilities of incorrect primary homologies. Three jackknife runs were undertaken, each with a different percentage deletion setting: 50% (40 characters), 36.25% (29 characters) and 10% (8 characters). The first and last values were chosen simply as large and small options respectively. The middle value was used to ‘emulate’ bootstrapping. Monte Carlo simulations showed that it is most likely that 29 characters will be duplicates in any given draw of 80 characters from a set of 80. Jack- knife calculations also take a very long time and so the number of random initializations for every replicate was set to 10. The number of replicates were 80, 115 and 1000 for the three runs in the order given above.

14 4. Results and Discussion G. illyricus in Britain

Figure 4: Collection locations for all 42 specimens that produced sequences for both trnLF and psbAtrnH.

4 Results and Discussion

The total number of specimens that provided sequence data was 49, with 42 successfully giving sequences for both trnLF and psbAtrnH. Only the latter were analyzed for this study: the rest are ignored for the rest of this discussion. All eleven specimens from the New Forest presented identical sequences for the two regions, suggesting that they are chloroplast clones as one might expect of a population on the edge of its species’ range (i.e. either founder effects after jump dispersal or progressive lineage sorting during leading edge migration). These eleven are treated here as a single entity here. The pan-Mediterranean distribution of the specimens under consideration is shown in Figure 4. Although the geographic area covered extends into northern Africa and the Middle East, the group of plants studied in this project will, in the great Eurovision tradi- tion, all be referred to as European, mainly for convenience when comparisons are made to the southern African species. The 42 sequences grouped together to give 16 unique haplotypes. The groupings are listed in Figure 6. One of the first observations is the lack of any strong correlation between species identification and haplotype. This could be indicative of significant taxonomic problems for this group. This will be discussed later; in the meantime the species determinations will be ignored and instead the haplotype groupings will be referred to as the ordinary taxonomic units representing the European species.

15 4. Results and Discussion G. illyricus in Britain

a b c d e f g h i j k l m n o p q A C A - - T - - G G - - - - - T G T ------T GT CT CA - G G - G B C A - - T - - G G - - - - - T G T ------T GT CT CA A G G - G C C A - - T A T G G - - - - - T G T ------T GT CT CA A G G - G D C A - - T A T T G - - - - - T T C ------T GT CT CA A G G - G E C A - - T A T T G - - - - - T T T ------T GT CT CA A G G - G F C A - - T A T T G - - - - - T T T ------T GT CT CA A G T - G G C A A - T A T G G - - - - - T G T ------T GT CT CA A G G - G H C G - - C A T G G - - - - - T G T ------T GT CT CA A G T - G I C G - - T A T G G - - - - - T G T A A T G T A T GT CT CA A G T - G J T A - - T A T G G - - - - - T G T ------C TG AG AC A A G - G K T A - - T A T G T - - - - - T G T ------T GT CT CA A G G - G L T A - - T A T G T - - - - - T G T ------T GT CT CA A G G T G M T A - - T A T T G C T C T T T G T ------T GT CT CA A G G - C N T A A - T A T G G - - - - - C G T ------T GT CT CA A G G - G O T A A - T A T G G - - - - - T G T ------T GT CT CA A G G - G P T A A A T A T G G - - - - - T G T ------T GT CT CA A G G - G

··· ··· 88 99 100 216 321 341 368 669 673 687 717 838 876 881 905 915 921 927 931 938 1195 1274 1320

Figure 5: The differences between the sixteen haplotypes arising for the 42 specimens that were fully sequenced. Each has been given a name in the form of a sans-serif letter and assigned a coloured symbol. The variable sites have been given lower-case letter designations. Thick diamonds are used for positions in trnLF; thin diamonds for psbAtrnH. Numbers refer to the position in the full alignment (Appendix G). Note: the reverse compliment region in J encompasses all the positions from 905 to 938.

A B C D RH13–G. italicus RH22–G. communis RH47–G. communis RH20–G. communis RH42b–G. italicus RH29–G. illyricus RH38–G. italicus

E F G H NF* –G. illyricus RH19–G. italicus RH35–G. illyricus RH33–G. illyricus RH06–G. communis RH46–G. communis RH60–G. italicus RH34–G. illyricus RH37–G. illyricus RH58–G. triphyllos

I J K L RH49–G. atroviolaceus RH42a–G. illyricus RH23–G. communis RH50–G. atroviolaceus RH51–G. atroviolaceus RH56–G. atroviolaceus

M N O P RH27–G. communis RH43–G. italicus RH52–G. anatolicus RH53–G. italicus RH44–G. italicus RH39–G. italicus RH54–G. italicus RH45–G. italicus RH59–G. italicus

Figure 6: Haplotype groupings for the 42 fully sequenced specimens. All specimens in a group have identical genetic sequences for the two marker regions. Refer to Figure 9 for the corresponding full haplotype relationship network.

16 4. Results and Discussion G. illyricus in Britain

Sub-family Relationships

The first analysis considered the relationship of the European species sampled to African Gladiolus and other closely related genera in the Iridaceae family, i.e. those of the sub- families Ixioideae (of which Gladiolus is a member) and the most closely related sub- family, Nivenioideae (containing the outgroup). The resulting phylogeny is presented in Figure 7. The corresponding trees given in Reeves et al. (2001)) are presented in Figure 8. Important in this study are the branches separating the two sub-families, the branches separating the Gladiolus clade from the rest of Ixioideae and the branches separating the southern African Gladiolus species used (G. guenzii) from the European species. The tree recovered agrees very well with the corresponding tree presented in the Reeves et al. (2001) paper. The differences can be attributed to possibly a) the inclu- sion of different taxa and that b) a slightly different (and presumably slightly smaller) subset of characters was used here. However, the fact that our treatment of characters will have differed adds weight to the congruent topology. The only direct conflicts are the relationships within the Geissorhiza–Hesperantha–Schizostylis clade and within the monophyletic Babiana–Chasmanthe–Ixia–Sparaxis–Tritonia group. In all important aspects (as defined above), the trees agree exactly; namely, in that Ixioideae is monophyletic and Gladiolus is the sister to the rest of the sub-family. The former is very strongly supported: Ixioideae appears as a single group, underpinned by twelve synapomorphic homologies and high statistics values. On the other hand, the latter observation is very weak. Gladio- lus as the sister to the rest of Ixioideae is not well supported in either treatment. Here, it is supported by two character changes: one is ambiguous (relying on ACCTRAN optimization), but uniquely synapomorphic; and the other is consistent and unambiguous, but is homoplastic (CI=0.5, RI=0.9) with an autapomorphic change for Nivenia in the outgroup sub-family. Given the strength of the separation between the ingroup and outgroup taxa, this last homoplasy relation can actually be overlooked, meaning that a definite synapomorphy holds Gladiolus as the sister genus in the trnLF region. However, being reliant on a single character such a conclusion remains tenuous. This point is emphasized by the tree Reeves et al. (2001) present as the result of their four plastid combined analysis, which gives Watsonia and Lapeirousia as the basal sister group in Ixioideae. Their conclusion on this matter is equally weak, with no significant statistical support. Otherwise, their combined data tree is also in broad agreement with the other two, in all revelent aspects. The consistent signal is that Gladiolus is a distinct member of the sub-family Ixioideae (less related to any other genera in the rest of sub-family than any other is to their closest relation) and an individually paraphyletic taxon (the smallest clade including it being

17 4. Results and Discussion G. illyricus in Britain

Chasmanthe aethiopica (1) (1) Babiana eckionii (1) (2) Tritonia disticha [;;95] Ixia latifolia (2) Sparaxis variegata [;;99] (2) Geissorhiza heterostyla (3) [;;97] Hesperantha pseudopilosa [;92;100] Schizostylis coccinea Radinosiphon leptostachya (2) Crocus puichellus [;;98] Syringodea bifucata 1 (1) Romulea monadelpha [;;100] (1) Lapeirousia neglecta Ixioideae (1) Tritoniopsis unguicularis Savannosiphon euryphyllus (2) Freesia alba [;;96] Anomatheca laxa (2) Micranthus junceus 12 (4) [;;97] (1) Thereianthus reacemosus 92 [91;96;100] Watsonia angusta Pillansia templemannii 1 (1) A ,B C ,D ,E ,F ,G ,H ,I (3) 3 (3) N 97 [90;97;100] 94 [94;98;100] 5 (5) J ,M ,O ,P 94 [94;98;100] K ,L Gladiolus guenzii (3) Klattia ﬂava (2) 98 [;99;100] Witsenia maura [;;97] Nivenia corymbosa Aristea coerules Geosiris aphylla

Patersonia glabrata Nivenioideae

Figure 7: The strict consensus of the most parsimonious trees found for this study, showing the cladistic relationships between the genera of the sub-families Ixioideae and Nivenioideae. Below branches is the bootstrap support (%), followed by jackknife values in square brackets (given in descending order of deleted-character count, i.e. 50%, 36.25% & 10%). Values below 90% have been omitted. Above all internal branches the Bremmer support is in parentheses. Selected branches have the minimum branch length given in front of the Bremmer support. The European species are represented here by their haplotype group letters and corresponding coloured symbols. The relationship between the letters, symbols and actual specimens is given in Figure 6 on page 16. Although the species outside Gladiolus have been used to represent their genus, their full names have been used as a reminder of the error potential remaining in sampling artefacts. This tree is based on trnLF only.

18 4. Results and Discussion G. illyricus in Britain

3 Chasmanthe 4 Chasmanthe 1 Babiana 4 Ixia 3 Ixia 5 Babiana 1 Tritonia 2 Tritonia 4 Sparaxis 1 Sparaxis

5 Geissorhiza 3 1 Crocus 2 94 1 Hesperantha Romulea Schizostylis 6 Freesia 78 1 Radinosiphon Anomatheca

2 Crocus 21 Geissorhiza 56 Syringodea 99 12 Hesperantha 6 83 Romulea 1 Schizostylis 3 7 4 Lapeirousia Syringodea 1 Tritoniopsis 7 Radinosiphon Savannosiphon 6 Savannosiphon 2 1 3 Freesia Tritoniopsis 53 Anomatheca 7 Micranthus 97 2 Micranthus 4 Thereianthus 26 82 22 65 1 Thereianthus 100 Pillansia 93 Watsonia 22 Gladiolus

11 Pillansia 19 15 Lapeirousia 7 88 Gladiolus 99 100 Watsonia 9 32 11 Klattia 23 Klattia 8 99 16 100 75 Witsenia 95 Witsenia 90 100 11 Nivenia 22 Nivenia 90 20 Aristea 95 20 Aristea 31 Geosiris 86 Geosiris 33 Patersonia 63 Patersonia

Figure 8: Two trees from Reeves et al. (2001) for the genera of Ixioideae and Nivenioideae. Left: result using only trnLF. Right: a tree using all four of the plastid regions employed in their study. Branch lengths (ACCTRAN optimization) are given above the branches. Bootstrap values above 50% are given below the branches. Note: the absolute branch lengths are not comparable across trees because different data matrices were used for each. equal or close to the entire subfamily). Based on a crude molecular-clock inspection, it would seem that Gladiolus was split from the rest of the sub-family by one of the ﬁrst speciation events, but the exact basal relationships are very ambiguous using only these limited plastid data. Concentrating on the phylogeny created for this study, it can be seen that Gladiolus appears as a coherent monophyletic group, agreeing with the well established, morphologically based taxonomy. For the strict consensus tree, the Gladiolus clade is deﬁned by four unique (CI=1.0, RI=1.0) character changes and one ‘slightly’ homoplastic character transition (CI=0.5, RI=0.833∗) shared with an autapomorphy for Micranthus. In addition, there is very high statistical support for this clade. Finally, the distinctiveness of the European Gladiolus taxa within the Gladiolus grouping is also solid. They are separated from the southern African species by six character transitions, all of which are unique within inside the Gladiolus clade (albeit not so across all taxa: CI=0.2–0.667, RI=0.0–0.860∗). The grouping of the European sequences also has high bootstrap and jackknife support. Obviously, with only one species representing the entirety of the sub-Saharan Gladioli, it is impossible to say whether this would form

∗Reticulation index values are possibly inﬂated by the excess of Gladiolus taxa in the data matrix.

19 4. Results and Discussion G. illyricus in Britain

Key: I intra-region homoplasy

l inter-region homoplasy trnLF change id H d J psbAtrnH change id b

F f j m K p L

o o g

D k E f j C a q f h M

c c e

B G a O c2 P

i n

A N

Colour Key: west of Italy predominately west of Italy east of Italy

Figure 9: Haplotype network: all the shortest evolutionary haplotype pathways are represented. The most ancient haplotype, determined by comparison to the outgroup, G. guenzii, is shown as a ﬁlled black circle. The group containing the New Forest species has a double circle. a sister group to the European clade as seen here, whether they would form a pectinate ladder of derivations from the most ancestral leading to the northern species, or whether the relationships between global Gladiolus species is much more interleaved. Given the geographic distances involved, plus the seemingly rapid pollinator-mediated radiation of the southern African species (Goldblatt, 2001), the last option would be very unlikely, while the ﬁrst possibility would be most probable.

20 4. Results and Discussion G. illyricus in Britain

h A e B

C & G

k D a f j E

o F

d H b o l I

m J

K g p L

f h q M

i N

O & P

G. guenzii

Figure 10: Cladogram for the haplotypes of this study, after resolution of reticulations.

Relationships within Europe

Focussing back on the more central issue of the relationships between the European species in isolation, consideration was given to the variety of haplotypes encountered. The network for the specimens sequenced in this project is in Figure 9. Homoplasy appears in three places: in the reticulated ambiguity of a and c ; the ambiguity of f , j and o ; and the unambiguous parallel evolution of f for the M grouping. The two ambiguities were resolved in different ways. The ﬁrst involved c , which is the end of the homopolymer near the start of the trnLF region. As discussed in the previous section, this is a high homoplasy potential site, meaning that parallel evolution is more likely than

21 4. Results and Discussion G. illyricus in Britain expected for most indel changes. As such there is little chance of guessing the correct evolutionary event sequence and so this position was ignored. The result was the grouping of G with C (effectively assuming parallel evolution) and of O with P , the latter satisfactorily being biogeographically plausible. The other ambiguity naturally presented four alternatives; two involving homoplasy in f + j and two for o . Based on the parsimony criterion, the former can be rejected, leaving the choice between parallel evolution or a reversal at site o . Assuming a reversal has occurred (i.e. removing the branches between G and E ) results in a fully resolved tree for the ﬁrst nine groups (A to I ). However, assuming parallel evolution is more desirable for two reasons: i) it generates a shallower tree and so assumes a lower rate of change, which could be considered more parsimonious, and ii) its tree is also the strict consensus of the two alternatives. Using these considerations, and using G. guenzii as the outgroup, a rooted cladogram was constructed, as shown in Figure 10. The cladogram highlights the fact that most of the variation observed was autapomorphic, with few clades held together by more than one character, making cladistic analysis rather inappropriate. Therefore, analysis will continue to be based on viewing the data from a haplotype network perspective, i.e. regarding links as having a direct relation to ancestry. The relationships between the European taxa was used to inspect the biogeography of the sampled specimens. Four specimens were removed at this stage to simplify the analysis, all appearing far from the expected range of the haplotype group at which they were assigned:

RH06 (C ): most likely to be a UK garden escapee, although the determination as it being a different species to the rest of its assigned grouping (found around central Medi- terranean) is supported by its morphology and haplotype. However, its genetic difference is rooted on the problematic homopolymeric site at c , making it a wildcard specimen that is justiﬁably ignored.

The other three can be grouped together as a set of trans-Mediterranean anomalies:

RH58 (D ): very interesting as it has the same haplotype as the New Forest plants and the Iberian G. illyricus specimens, but was collected in Cyprus and has been determined to be a different species, G. triphyllos. RH59 (N ): identical to the rest of its otherwise Moroccan group in both haplotype and species determination, this specimen was found on Crete. RH60 (E ): also collected in Crete, with a determination of G. italicus, it has the same haplotype as what appears to be a G. illyricus plant in Portugal.

22 4. Results and Discussion G. illyricus in Britain

Figure 11: Distribution of the taxa that were fully sequenced, with lines connecting those with identical or nearly identical haplotypes. Dashed lines have been drawn between the closest locations in groups adjacent in the cladogram of Figure 10.

All three link the western tip of the Mediterranean to eastern Mediterranean islands. Taken together, they suggest that there could be an artificial conduit for transplantation in existence, especially as there are no geographically intermediate examples (although sampling of the intermediate area is sparse for this study). It is intriguing that all three were found on islands, suggesting possible isolation explanations, but being straddled on the mainland to both the north and south by the most ancestral haplotype of those sampled, human-mediated introduction seems most plausible, for at least RH59 and RH60, the two specimens collected on Crete. The specimen from Cyprus, RH58, is almost cer- tainly G. triphyllos, considered to be an endemic of the island. Indeed, Meikle (1985) believes that it has not been found anywhere else. Lack of entries in the floras of the surrounding countries support this. Morphologically, it is a smaller form of G. illyricus, but with flowers of a similar size and much more slender (proportionally thinner) leaves. Environmental conditions are an unlikely explanation for the difference with G. illyricus, because it shares some of its habitats with the local G. italicus. From its exclusive Cypriot island distribution (separated from the mainland for over 10 million years (Par- makelis et al. 2006)), its similar yet possibly distinct morphology and its shared genetics, together seem eminently indicative of classic, post dispersal, allopatric speciation by random drift. However, with only one specimen and no morphological or ecological data, no conclusions can be drawn. Therefore, these three species have all been removed from the analysis. Without the above anomalies, an apparent east–west genetic distinctiveness appears.

23 4. Results and Discussion G. illyricus in Britain

The haplotypes break down into roughly four groups:

A B D E O K L C F P M H I

West Mediterranean Central Med. East Med. Middle East

The above network is a collapsed version of the full haplotype network and so directly represents a projection of genetic similarity, i.e. the cladogram of Figure 10. The geographic ranges of each group have been added to show the congruence of this grouping with physical distribution. The one exception, which has been omitted from the groups above, is N , which is genetically more similar to O and P than it is it to C , despite being found in Morocco and Portugal. This aside, the correlation is signiﬁcant because C and O are most closely related to the sub-Saharan outgroup. Giving a high weight to distance over water, i.e. looking at temporal dispersal contours with a low probability on jump events, these two also have the largest ranges, as one might expect from the most ancestral haplotypes (Posada and Crandall, 2001). Therefore, even for the small chloroplast genome segment used here, there could be a signal indicative of the colonization history of the northern hemisphere by African Gladiolus.

Taxonomy

Comparing the species determinations for the haplotype group members from Figure 6 to their geographical distribution depicted in Figure 11, it is hard to discern much order. Little needs to be said of G. atroviolaceus (groups K and L ), being consistently separate in all of its geography, genetics and morphology. The focus is directed to the apparent interwoven relationship of G. italicus, G. communis and G. illyricus. None of these species occupy a single haplotype or geographic region, and no region or group holds a single species. At ﬁrst glance, N looks as though it might represent a strong G. italicus haplotype, but three of the specimens in this group were sampled from effectively the same location, simply making this an artifact of over-sampling. However, only one of this group is not from Morocco and is also the only member to not be identiﬁed as G. italicus. This ‘rogue’ sample is from Portugal. Portugal is also where the G. italicus exception in D was found. These observations begin to suggest that Portugal, or perhaps the glacial refugia of Iberia as a whole, could be a zone of high hybridization. On the other

24 4. Results and Discussion G. illyricus in Britain hand, group C , perhaps one of the most ancient haplotypes, is the only one to have its specimens identified as all three species (G. illyricus, G. communis and G. italicus), so it is possible that reticulation events have been spread throughout the Mediterranean since Gladiolus arrived. In any case, the genetic confusion seen here, even if only based on a small region of a plastid genome alone, is often reflected in the field (Lockton, 2006). Misidentifications are frequently mentioned in the Floras (e.g. Komarov, 1968) as well as the proliferation of hybridization, even as far east as to be seen between G. italicus and G. atroviolaceus (Feinbrun-Dothan, 1986). From existing texts, it could be suspected that chloroplast markers might not hold the answers: the majority of differentiating characters are floral or based on overall size. The former is commonly related to accelerated pollinator-mediated evolution, genetically manifest in the nuclear genome. The latter is often linked to ploidy level—a significant issue in the three species. Chromosome counts relating to the British G. illyricus have now become too confused to be reliable, so the relationships at the this level can not be commented upon. What is clear is that a resolution to this problem requires much denser sampling in a planned stratified manner, accompanied by ecological and cytological evidence.

25 5. Conclusions G. illyricus in Britain

5 Conclusions

This genetic project sheds light on the place of British G. illyricus in a broad European context, illuminating many of the issues that will need to be addressed by the further molecular investigations required to satisfactorily solve to the britannicus conundrum. This initial look at the chloroplast evidence strongly supports the existing notion of a monophyletic Gladiolus group, spanning the Sahara desert. The inspection of two chloroplast markers within the set of European specimens is less directly informative. It can be said that all the evidence gathered during this study supports the hypothesis that Gladiolus in the UK is a speciating archeophyte worthy of sub-species status, but mostly in the pas- sive sense of not contradicting it. However, from a Bayesian viewpoint, non-contradicting evidence is still positive evidence. It does seem clear that the plants in the New Forest came out of Iberia. The resolution available from trnLF and psbAtrnH does not allow for any more ﬁne-grained conclusions. However, many important points are raised and the range of the possible interpretations is a strong indicator for the direction of future investigation. The main issue for the next molecular study could well be ﬁnding a suitable region of the genome that can in some way consistently differentiate meaningful groups of sub-populations. From what was seen here, namely the little variation across the samples with little coherent ordering, chloroplast markers do not seem the good candidates. The trnLF and psbAtrnH regions are not suitable as barcoding markers for these species. Chloroplast capture, where, for example, lineage sorting of a hybrid population returns the phenotype to that of the paternal species (Tsitrone, 2003) could be a confounding factor. Furthermore, it is not known whether paternal chloroplast inheritance (Birky, 1995; Korpelainen, 2004) is possible in Gladi- olus species. The gene trees for nuclear and plastid gene can be very different, so these factors must be accounted for. The possible interrelatedness of the western Mediterranean populations in particular mean that it might be best to approach the matter from a population genetic standpoint rather than imposing the assumptions made by species-based phylogenetic analyses. Naturally, denser sampling is vital and so focussed hypotheses are needed to inform sampling strategy. Having determined the most likely source of the British plants, the route from the Iberian peninsula to Britain should be targetted to discover a) a more detailed picture of the parentage of the New Forest population and b) whether the migration pattern is evident and correlated with plausible natural rates. With few, or perhaps no locations for G. illyricus in France, there is pressure to investigate this as soon as possible.

26 5. Conclusions G. illyricus in Britain

A very important factor that must be considered is the ploidy level of all the populations involved. The base chromosome number for Gladiolus is thought to be one of the genus’ apomorphic traits, being x =15 (Goldblatt, 2001). Chromosome counts have been undertaken for European Gladiolus (van Raamsdonk and de Vries, 1989; Stokes, 1987), but these have become confused and so a new survey is required. Ploidy events are not reﬂected in the chloroplast relationships and so what was seen in this study could indeed be indicative of a ploidy-level based species concept in this group. It seems plausible, for example, that G. communis is a polyploid descendant of G. illyricus and that G. italicus is a polyploid hybrid with, perhaps, one of the eastern species. However, without ﬁrm cytological evidence, hypotheses at this stage will be mostly based on morphology, which as has been seen here to be possibly highly homoplastic, unless the species boundaries are very blurred meaning that some of the genetic variation is actually polymorphism. Morphology can be an excellent short-cut to genetic character sets, although one has to be careful when the underlying sets are large, dominant, polymorphic, etc. It was hoped that morphology could have been part of this project, but fresh samples from anywhere other than the New Forest could not be attained this year. Herbarium specimens can not be relied upon for morphological measurements because the same mechanism that is behind plant cells’ elongation when they expand can be responsible for distortions of length ratios during desiccation. Optimal growing conditions can also be indicative of genotype. If transplant exper- iments showed there to be a difference between the plants from the UK and those most closely related in Spain, then conservation status would be vital. Of course, knowing which plants to select outside of the British Isles relies on more informative genetic data. Here, the two markers used are part of the same linkage group. Evidence from several linkage groups is necessary for high resolution discrimination, particularly given the large range of difference inheritance mechanisms that could be in play. In conclusion, I would suggest that G. illyricus of Britain should remain a Schedule 8 species and be given a higher conservation priority, if not its own sub-species determination before more solid evidence is uncovered. The study of G. illyricus in the New Forest is the study of a fascinating situation, no matter which alternative turns out to be responsible for its presence here. However, the least interesting scenario would be an investigation of its decline to extinction.

27 Appendices G. illyricus in Britain

Figure 12: The typical June bracken covering of the habitat of G. illyricus in the New Forest. Photograph: Aeron Buchanan, Little Early (north), New Forest.

Acknowledgements

I would like to thank Fred Rumsey for taking me on as a student and gently guiding me in the right direction: this was a fantastic experience. I would also very much like to thank the following for their kind helpful and instruction: Steve Russell and his patient and straightforward tuition and insight in all aspects of laboratorical matters; Steve Ansell for sharing his in-depth knowledge of the ways of biogeography and his general, yet excellent tutorage; Harald Schneider for directing my thoughts in new directions, particularly on phylogenetic matters; Michael Grundmann for his friendly assistance whenever I needed it; and the sequencing laboratory for being so accommodat- ing.

This work was generously funded by • The Botanical Society of the British Isles, • The Botanical Research Fund and • Imperial College, London.

28 Appendices G. illyricus in Britain

A New Forest Specimens

Specimen Location OS grid ref. GPS State Extraction trnLF trnCD psbAtrnH ITS

NF01 D.W. SU34070585 N50◦ 510 5.4000 W01◦ 310 2.2800 fl. P25 () – NF02 D.W. SU34070585 N50◦ 510 5.4000 W01◦ 310 2.2800 fl. M64 – – NF03 D.W. SU34070585 N50◦ 510 5.4000 W01◦ 310 2.2800 veg. P26 – NF04 D.W. SU34060582 N50◦ 510 5.1600 W01◦ 310 2.4600 fl. NF05 B.W.P. SU34960486 N50◦ 500 32.6400 W01◦ 300 17.4600 veg. NF06 B.W.P. SU34960486 N50◦ 500 32.6400 W01◦ 300 17.4600 veg. M05 – NF07 B.W.P. SU34960486 N50◦ 500 32.6400 W01◦ 300 17.4600 veg.* NF08 B.W.P. SU34960486 N50◦ 500 32.6400 W01◦ 300 17.4600 veg. M65 – – – – NF09 B.W.P. SU34960486 N50◦ 500 32.6400 W01◦ 300 17.4600 veg. NF10 L.C. SU24811046 N50◦ 530 36.5400 W01◦ 380 53.7000 veg. P27 () – NF11 L.C. SU24811046 N50◦ 530 36.5400 W01◦ 380 53.7000 veg. NF12 T.K. SU26630888 N50◦ 520 48.9600 W01◦ 370 22.1400 veg. NF13 T.K. SU26630888 N50◦ 520 48.9600 W01◦ 370 22.1400 veg. M06 () – NF14 T.K. SU26630888 N50◦ 520 48.9600 W01◦ 370 22.1400 veg. NF15 T.K. SU26630888 N50◦ 520 48.9600 W01◦ 370 22.1400 veg. M66 – – – – NF16 T.K. SU26630888 N50◦ 520 48.9600 W01◦ 370 22.1400 veg.* NF17 K.S. SU26830642 N50◦ 510 24.7200 W01◦ 370 11.1600 veg. M67 – – – – NF18 K.S. SU26830642 N50◦ 510 24.7200 W01◦ 370 11.1600 veg. NF19 K.S. SU26830642 N50◦ 510 24.7200 W01◦ 370 11.1600 veg. NF20 K.S. SU26830642 N50◦ 510 24.7200 W01◦ 370 11.1600 veg. P19, P20 () () NF21 K.S. SU26830642 N50◦ 510 24.7200 W01◦ 370 11.1600 veg. NF22 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 fl. P02 () – NF23 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 fl. NF24 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 fl. NF25 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 fl. NF26 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 veg. NF27 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 veg. NF28 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 veg. NF29 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 veg. M07 () – NF30 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 veg. NF31 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 veg. NF32 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 fl. M68 – – – – NF33 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 fl. NF34 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 veg. NF35 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 fl. NF36 L.E. SU22630411 N50◦ 500 9.800 W01◦ 400 47.400 fl. NF40 C.B. SU23370109 N50◦ 480 32.600 W01◦ 400 10.800 veg. NF41 C.B. SU23370109 N50◦ 480 32.600 W01◦ 400 10.800 veg. NF42 C.B. SU23340116 N50◦ 480 33.000 W01◦ 400 11.100 veg. NF43 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 fl. NF44 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 veg. NF45 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 veg. NF46 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 fl. M08 – NF47 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 veg. NF48 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 veg. P01 () () NF49 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 fl. NF50 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 veg. NF51 C.B. SU23430112 N50◦ 480 32.800 W01◦ 400 9.200 fl. NF52 Y.B. SU25690032 N50◦ 480 11.3400 W01◦ 380 11.4600 fl. P28 () –

Key: = Sequencing successful () = PCR successful fl. = flowering plant = PCR unsuccessful – = not attempted veg. = non-flowering plant *whole plant collected

Locations: D.W.=Denny Wood; B.W.P.=Bishop of Winchester’s Purlieu (Woodﬁdley Passage); L.C.=Lucas Castle; T.K.=The Knowles; K.S.=Warwick Slade; L.E.=Little Early; C.B.=Clayhill Bottom; Y.B.=Yewtree Bottom.

Specimens: held by Fred Rumnsey, Botany Department, Natural History Museum. Extractions: held by Steve Russell, Botany Cell and Molecular Laboratory, Natural History Museum.

29 Appendices G. illyricus in Britain

B Reading University Herbarium Specimens

Specimen Species* Location Coll. Date Identiﬁer Notes* Extraction trnLF trnCD psbAtrnH ITS

RH01 G. illyricus UK 1960-06-21 M09,P13 – RH02 G. illyricus UK 1950-06-18 P70 – – RH03 Gladiolus Scilly 1938-06-01 “227” G. communis? RH04 G. communis Guernsey 1958-05 “681” G. italicus? P75 – – RH05 G. communis Scilly 1957-05-26 “768” G. italicus? RH06 G. communis UK 1982-05-30 “1283” P43 – – RH07 G. illyricus UK 1982-05-30 “1283” G. communis P51 – – RH08 G. communis UK 1984-05 “3310” P46 – – RH09 G. italicus Portugal 1967-04-13 P00 25516 BRAHMS; G. illyricus RH10 G. italicus France? 1974-04-16 P00 25515 BRAHMS P61 – – RH11 G. italicus Portugal 1967-04-01 P74 – – RH12 G. italicus Portugal 1967-04-07 RH13 G. italicus Malta 1985-03 P00 25520 BRAHMS P47 – – RH14 G. italicus Greece 1978-03 P00 25521 BRAHMS; G. communis? P55 – – RH15 G. italicus Greece 1979-04 P00 25512 BRAHMS M10,P14 – RH16 G. italicus Spain 1973-04-22 P00 25508 BRAHMS P76 – – RH17 G. italicus Spain 1979-05-17 P00 25520 BRAHMS P60 – – RH18 G. italicus Spain 1952-05-22 “1994” RH19 G. italicus Portugal 1981-04-17 “13701” P59 – – RH20 G. communis Spain 2005-04-15 P00 23549 P21 – – RH21 G. communis Mallorca 1989-05 P52 – – RH22 G. communis Bulgaria 1999-05-31 P00 08227 P04 – RH23 G. communis Greece 1993-05-24 “245” P29 – RH24 G. palustris Germany? 1902-07-06 RH25 G. imbricatus Poland? 1976-07-13 “682” P58 – – RH26a G. imbricatus Russia? 1974-07-05 “I27” P62 – – RH26b G. illyricus Portugal 1980-07-20 “879” P53 – – RH27 G. illyricus Portugal 1980-04-18 “10937” G. communis M11,P15 – RH28 G. illyricus Portugal 1986-04 “4398” G. communis? P42 – – RH29 G. illyricus Menorca 2004-04-25 P00 18444 M12,P16 RH30 G. illyricus Italy 1997-06-13 “1379” G. communis? P30 – RH31 G. illyricus Yugoslavia 1980-06-02 P45 – – RH32 G. illyricus Yugoslavia 1972-07 P69 – – RH33 G. illyricus Spain 1994-04-20 “163” P31 () RH34 G. illyricus Spain 1996-04-28 “17252” G. communis? P17,P18 – RH35 G. illyricus Portugal 1992-05-18 “8351” P32 – RH36 G. illyricus Spain 1988-04-10 “9463” RH37 G. illyricus Spain 1994-04-26 “751” P33 () – RH38 G. italicus Tunisia 1990-04-26 P00 25528 BRAHMS P03 – RH39 G. italicus Libya 1970-04-02 P00 25529 BRAHMS P72 – – RH40 G. communis Algeria 1976-04-13 “69” G. italicus? P56 – – RH41 G. communis Algeria 1976-04-15 “232” G. italicus? P57 – – RH42a G. illyricus Morocco 2004-06-12 P00 18556 P22 () – RH42b G. communis Morocco 1994-05-25 “3357/94” G. italicus P34 – RH43 G. italicus Morocco 1995-04-21 “16531” P35 – RH44 G. italicus Morocco 1994-04-40 P00 25524 BRAHMS P36 – RH45 G. italicus Morocco 1995-04-08 P00 25526 BRAHMS P37 – RH46 G. communis Morocco 2001-06-16 P00 02442 P23 – RH47 G. communis Morocco 1994-03-07 “14080” G. italicus? P38 – RH48 G. communis Morocco 1974-03-29 “157” G. italicus? P63 – – RH49 G. atroviolaceus U.S.S.R 1979-05-11 “9898” P54 – – RH50 G. atroviolaceus Israel 1989-03-23 “496” P44 – RH51 G. atroviolaceus Iran 1972-05-04 P00 03838 P73 – – RH52 G. anatolicus Turkey 1985-04 “3553” P48 – – RH53 G. italicus Lebanon 2000-04-18 P00 05208 P41 – – RH54 G. italicus Lebanon 2000-04-25 P00 05207 P50 – – RH55 G. italicus Jordan 1973-04-09 P00 25532 BRAHMS; G. atroviolaceus? RH56 G. atroviolaceus Iraq 1985-05-15 “93” P49 – – RH57 G. italicus Iraq 1958-04-01 “127” P71 – – RH58 G. triphyllos Cyprus 1997-05-10 P00 26826 P39 – RH59 G. italicus Crete 2003-06-06 P00 18466 BRAHMS P24 RH60 G. italicus Crete 1999-04-30 P00 02250 BRAHMS P40 –

Key: = Sequencing successful () = PCR successful = PCR unsuccessful – = not attempted *Species=det. on sheet; Notes=correction (or ‘?’ denotes suspected correction).

Specimens: held by Fred Rumnsey, Botany Department, Natural History Museum (including medium resolution sheet photographs). Extractions: held by Steve Russell, Botany Cell and Molecular Laboratory, Natural History Museum.

30 Appendices G. illyricus in Britain

C GenBank Sequences

Species Seq. Reference GenBank Accession (trnLF)

Iridaceae: Nivenioideae Aristea coerules Chase et al. 2000 AJ290285, AJ290319 Geosiris aphylla Goldblatt et al. 2001 AJ409616 Klattia ﬂava Goldblatt et al. 2001 AJ409617 Nivenia corymbosa Chase et al. 2000 AJ290287, AJ290321 Patersonia glabrata Chase et al. 2000 AJ290284, AJ290318 Witsenia maura Chase et al. 2000 AJ290286, AJ290320

Iridaceae: Ixioideae Anomatheca laxa Goldblatt et al. 2001 AJ409575 Babiana eckionii Goldblatt et al. 2001 AJ409586 Chasmanthe aethiopica Goldblatt et al. 2001 AJ409572 Crocus puichellus Goldblatt et al. 2001 AJ409580 Geissorhiza heterostyla Goldblatt et al. 2001 AJ409589 Gladiolus guenzii Goldblatt et al. 2001 AJ409574 Hesperantha pseudopilosa Goldblatt et al. 2001 AJ409579 Ixia latifolia Goldblatt et al. 2001 AJ409581 Lapeirousia neglecta Goldblatt et al. 2001 AJ409567 Micranthus junceus Goldblatt et al. 2001 AJ409570 Pillansia templemannii Goldblatt et al. 2001 AJ409585 Radinosiphon leptostachya Goldblatt et al. 2001 AJ409573 Romulea monadelpha Goldblatt et al. 2001 AJ409576 Savannosiphon euryphyllus Goldblatt et al. 2001 AJ409568 Schizostylis coccinea Goldblatt et al. 2001 AJ409578 Sparaxis variegata Goldblatt et al. 2001 AJ409582 Syringodea bifucata Goldblatt et al. 2001 AJ409584 Thereianthus reacemosus Goldblatt et al. 2001 AJ409569 Tritonia disticha Goldblatt et al. 2001 AJ409588 Tritoniopsis unguicularis Goldblatt et al. 2001 AJ409577 Watsonia angusta Goldblatt et al. 2001 AJ409566

31 Appendices G. illyricus in Britain

D Total DNA Extraction Protocol

Total DNA Extraction This is a near-verbatim copy of Karen James’ modiﬁcations to Steve Russell’s adaptation of Rogers and Bendich (1994). The instructions that were not undertaken have been omitted without com- ment. Some notes have been included, commenting on variations and their success.

Preparation: Acid-wash pestles and mortars.

Grind plant material: 20-50mg leaf in pestle and mortar with equal volume of acid-washed sand. Alternatively, grind by machine with acid-washed bead [grinding by machine resulted in much reduced yields, fatally so for herbarium material].

Extraction: • Add 500µl CTAB buffer. • Add 50µl of SARKOSYL. • Add 10µl (10mg/ml) PROTEINASE K. • Mix vigorously by vortex and leave for 30–60 minutes in 60◦C heat block with occasional vortexing.

SEVAC extraction: Do twice • Add an equal volume of SEVAC (roughly 500µl). • Mix vigorously by vortex and centrifuge (top speed for 3 minutes). • Pipette off the clear supernatant (DNA) layer, without disturbing the white in- terface layer, into a fresh eppendorf tube.

Precipitation: • Add 2/3 volume (roughly 400µl) ice cold ISO-PROPANOL. • Mix by inversion and leave on ice for 30 minutes. • Centrifuge (top speed for 3 minutes) [DNA pellets should appear]. • Remove ISO-PROPANOL. • Wash brieﬂy with 500µl of 70% ethanol by inversion. • Centrifuge (top speed for 3 minutes). • Remove ETHANOL and dry in 60◦C heat block with lid open. • Re-suspend pellet in 15µl DISTILLED WATER and wait until dissolved. • Proceed to puriﬁcation

Alternative precipitation: this was tried for a small number of samples and was found to precipitate polysaccharides and other goo too readily and in some cases, excessively. • Add 2/3 volume (roughly 400µl) ice cold ISO-PROPANOL. • Mix by inversion and leave on ice for 30 minutes. • Centrifuge (top speed for 3 minutes) [DNA pellets should appear]. • Remove ISO-PROPANOL.

32 Appendices G. illyricus in Britain

• suspend pellet in 180µl 0.1M T.E.BUFFER + 20µl 3M SODIUM ACETATE; heat at 60◦C until dissolved. • Add 600µl ice cold 100% ETHANOL; mix by inversion. • Store at -20◦C for at least 1 hour. • Centrifuge at 4◦C for 3 minutes (returning tube to ice after centrifuging); remove ETHANOL. • Wash brieﬂy with 500µl of 70% ethanol by inversion. • Centrifuge (top speed for 3 minutes). • Remove ETHANOL and dry in 60◦C heat block with lid open. • Re-suspend pellet in 15µl DISTILLED WATER and wait until dissolved. • Proceed to puriﬁcation

DNA Solution Puriﬁcation

This is a near-verbatim copy of the “Purification of DNA from solution” protocol in the Amer- sham Biosciences GFX PCR DNA and Gel Band Purification Kit (product code: 27-9602-01) instructions booklet. This kit is now manufactured by G.E. Healthcare. The booklet has two unas- signed references: B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. USA 76, 615 (1979); M. A. Marko, R. Chipperfield and H. C. Birnboim, Anal. Biochem. 121, 382 (1982).

Purification 1. Place one GFX column in a column tube for each purification to be performed. 2. Add 500µl of CAPTURE BUFFER [blue top] to the GFX column. 3. Transfer the DNA solution (up to 100µl) to the GFX column. 4. Mix thoroughly by pipetting the sample up and down 4–6 times. 5. Centrifuge in a microcentrifuge at full speed for 30 seconds. 6. Discard the flow-through by emptying the collection tube [use a pipette]. Place the GFX column back inside the collection tube. 7. Add 500µl WASH BUFFER to the column. Centrifuge at full speed for 30 seconds. 8. Discard the collection tube and transfer the GFX column to a fresh 1.5ml microcentrifuge tube (i.e. NOT a collection tube). 9. Apply 50µl autoclaved double-distilled water directly to the top of the glass fibre matrix in the GFX column [making sure not to touch the matrix with the pipette tip]. 10. Incubate the sample at room temperature for 1 minute. 11. Centrifuge at full speed for 1 minute to recover the purified DNA.

Band extraction: The band is identiﬁed on the gel and removed carefully with a scalpel, taking as little excess agarose as possible. The extracted gel sample is dissolved in 10µl CAPTURE BUFFER for every 10mg of gel. After vigorous vortexing, the sample is left to incubate at 60◦C for 5–15min until completely dissolved. Puriﬁcation proceeds as above (from step 3).

33 Appendices G. illyricus in Britain

E Polymerase Chain Reactions

Reactions were carried out in two types of machine: Techne’s Techgene Thermal Cycler (http://www.techne.com/) with program 1 or 2 below; or Thermo Fisher Scientiﬁc, Inc’s Px2 Thermal Cycler (http://www.thermo.com/) with program 3 below. All gradient transitions were set to their maximum rates.

Programs 1. heated lid 109◦C; initial denaturing: 2min @ 94◦C; 30× { 30sec @ 94◦C; 30sec @ 50◦C; 2min @ 72◦C }; ﬁnal extention: 5min @ 72◦C 2. heated lid 109◦C; initial denaturing: 1min @ 94◦C; 30× { 1min @ 94◦C; 1min @ 54◦C; 3min @ 72◦C }; ﬁnal extention: 8min @ 72◦C 3. sim tube; stage 1.1: 2min @ 94◦C; 30× {2.1: 30sec @ 94◦C; 2.2: 30sec @ 50◦C; 2.3: 2min @ 72◦C }; 3.1: 5min @ 72◦C

Programs 1 and 3 were used for trnLF, trnCD and psbAtrnH. Program 2 was used for the ITS region. Each reaction was 25µl with components in the following ratio: 0.5µl TAQ enzyme, 1µl forward primer, 1µl reverse primer, 0.5µl nucleotide stock, 1.5µl Magnesium Chlo- ride solution, 2.5µl reaction buffer, plus enough DNA solution to make the mixture up to 25µl and provide roughly 100ng of template. For extractions where the yield was very low, excess DNA solution (of the order of 2 or 3µl) was added to achieve the 100ng target. This entire procedure must be carried out on ice. ‘Master Mixes’ for two or more (typically four) reactions were made together to make the pipetting more reliable.

34 Appendices G. illyricus in Britain

F Sequence Reactions

Reactions were carried out in two types of machine: Techne’s Techgene Thermal Cycler (http://www.techne.com/) with program 1 below; or Thermo Fisher Scientiﬁc, Inc’s Px2 Thermal Cycler (http://www.thermo.com/) with program 2 below. All gradient transitions were set to their maximum rates.

Programs 1. heated lid 109◦C; initial denaturing: 5min @ 96◦C; 25× { 10sec @ 96◦C; 5sec @ 50◦C; 4min @ 60◦C } 2. sim tube; stage 1.1: 1min @ 96◦C; 25× {2.1: 10sec @ 96◦C; 2.2: 5sec @ 50◦C; 2.3: 4min @ 60◦C }

Both programs were used for all regions. Each reaction was 10µl with components in the following ratio: 3µl sequencing buffer, 1µl 0.1× dilution primer, 1µl Big Dye, plus 5µl DNA solution, diluted to make the mixture contain between 2–3ng of PCR product per 100 base pairs of region being ampliﬁed. For the trnLF and psbAtrnH segments, roughly 15ng of product was incorpo- rated in each reaction mixture. For trnCD and ITS longer sequences were expected and so 22ng was the target. The entire procedure must be executed on ice. ‘Master Mixes’ for all reactions with a particular primer were made together to make the pipetting more reliable.

35 Appendices G. illyricus in Britain

G Alignments

The combined alignment of trnLF (1–782) and psbAtrnH (783–1443), with primer regions included. Polymorphic regions have been indicated at their start and end (except for singletons for which only the start is indicated) with the exact position number (within

this alignment) of the start of the region.

20 10 CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA CGAAATCGGTAGACGCTACGGACTTGATTGAATTGAGCCTTGGTATGGAAACCTGCTAAGTGGTAACTTCCAAATTCAGA NFr01 NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60

Appendices G. illyricus in Britain

230

220 216

TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA CGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA TGGTAGCCGGAATCTTTCTA

210

200

190

180

170

160

150

140

130

120 110

GGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGTGGGGCAATCCTGAGCCAAATCTTTATTTTGAGAAAACAAAAACTAGAATAAAAAAGGATAGGTGCAGAGACTCAATGGAAGCTGTTCTAACGAATGGAGTTGACTACGTTGTGT

100 99

A-- A-- A-- A-- A-- A-- A-- A-- A-- A-- A-- AA- A-- A-- A-- A-- A-- AA- A-- A-- A-- A-- A-- A-- AAA G-- A-- AA- AA- AA- G-- A-- A-- A-- A-- AA- A-- AA- A-- A-- AA- A--

90 88 CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA TGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA CGGAAAAAAAA CGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA TGGAAAAAAAA CGGAAAAAAAA TGGAAAAAAAA CGGAAAAAAAA GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC GAAACCC NFr01 NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60

Appendices G. illyricus in Britain

390

380

370 368

GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT TAATATTCGGTGATCAAATCTAT TAATATTCGGTGATCAAATCTAT TAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT TAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT GAATATTCGGTGATCAAATCTAT

360

350

341

340 TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC GTGGATCTATTCCAATCGAGAAGAATC TTGGATCTATTCCAATCGAGAAGAATC 330

GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT GCAAAATGGGGAGAAATT

321

320 AT AT AT AT AT AT AT AT AT AT AT AT AT AT -- AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT -- AT AT AT AT AT AT AT AT AT AT

310

300

290

280

270

260

250 240 TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT TCGAAATGAAAGATAAGGATATACCTAATTCGTATACATATTACCATATCAAACGATTAATCATGACCCAAATCATTATATATAT NFr01 NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60

Appendices G. illyricus in Britain

540

530

520

510

500

490

480

470

460

450

440

430

420

410 400 CATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAAGAGAGAGTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAAAAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCGGAAGAGAATAAA NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60 NFr01

Appendices G. illyricus in Britain

700

690 687

TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT CCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT CCCAAGTACTCTAT CCCAAGTACTCTAT CCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT TCCAAGTACTCTAT CCCAAGTACTCTAT TCCAAGTACTCTAT 680

TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA TAATTTCCATAGA

670 669

------CTCTT ------

660

650

640

630

620

610

600

590

580

570

560 550 CCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGATCTAAAACTCAGAAATTTTGGGGACTGTGCCAAATTTTTTAATACTTTTGTAGTCTCTTCCCCAATAAAAAGTCCATTTTACTTCCTAACAATTTCTATTATTCTATTCTTTTTGAATGAAGAT NFr01 NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60

Appendices G. illyricus in Britain

850

840 838

TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT CTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT TTCCATCTATAAATGGAT

830

820

810

800

790

780

770

760

750

740

730

720 717

TAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGTAAATAGTCGGGATAGCTCAGTTGGTAGAGCAGAGGACTGAAAATCCTCGTGTCACCAGTTCAAATGTTATGCATGAACGTAATGCTCACAACTTTCCTTTAGACCTAGCTGCTGTCGAAGGAAATAGTCGGGATAGCTCAGT 710 ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG ATAGGATGATGCGCGG NFr01 NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60

Appendices G. illyricus in Britain

1010

1000

990

980

970

960

950

940 938

GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA ACATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA GCATGGATCCCCCACCCTTGTTTGATCTATCCAAAATTAACGACGAGATTTATTATCGTTTCTCGCATGTCTA

931 930 ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG -TATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG ATATGGG

AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA 927 CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA AC CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA CA

AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAAA

921 920 CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT AG CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT CT

TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT 915

GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT TG GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT GT

910 905

TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT CCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT TCCCATATTT

900 890

TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG TACGAATCGTTGAAGGATCCATG

880 876

------AATGTA ------

870 860 AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA AAGATTTCCCTATTAATGTA NFr01 NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60

Appendices G. illyricus in Britain

1160

1150

1140

1130

1120

1110

1100

1090

1080

1070

1060

1050

1040

1030 1020 GCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATCTGTTATATAAATAGGCAAATGCTCCTTTCCATTATGAATAGCGATTGTATGGCCAATCATTGTGGGTATAATGGTAGATGCCCGAGACCAAGTTACTATTATGCGAAAGTCAAAGTAGGCGCGAATTCTCCCAATTTGTGACCTACCATACGATC NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60 NFr01

Appendices G. illyricus in Britain

1320 1320

G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G C G G G G G

1310

1300

1290

1280 1274

-ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC TACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC -ACATTTTAAAGATTGGCATTCTATGTCCAATAGAATATCTCGATC

1270

1260

1250

1240

1230

1220

1210

1200 1195

TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT TAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT GAATTTTTCCCGATAAATGATTGGCTACAAAAGGATTTTTTTTTAGTGAACGTGTCACAGCGGATTACTCCTTTTTTTT

1190

1180 1170 TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT TTCTTTCTCCTCCCTCATGTTGAGTTTTT NFr01 NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60

Appendices G. illyricus in Britain

1440

1430

1420

1410

1400

1390

1380

1370

1360

1350

1340 1330 AAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGAATCCACCATGCGCGAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAAAGAGAAAATCCTTTAGC NFr02 NFr03 NFr06 NFr10 NFr13 NFr20 NFr22a NFr29 NFr48 NFr52 RH06 RH13 RH19 RH20 RH22 RH23 RH27 RH29 RH33 RH34 RH35 RH37 RH38 RH39 RH42a RH42b RH43 RH44 RH45 RH46 RH47 RH49 RH50 RH51 RH52 RH53 RH54 RH56 RH58 RH59 RH60 NFr01

45 Appendices G. illyricus in Britain

Full alignment of the trnLF region for representative Gladiolus sequences and other genera of Ixioideae and Nivenioideae. The positions selected for cladistic analysis are highlighted: those excluded from the data matrix are grey. Gladiolus sequences that added no information within the characters used were omitted for efﬁciency. 10 20 30 40 50 60 70 80 90 100

New Forest GladiolusCGAAATCGGTAGACGCTACGGACTTGATTGAATTGA-----G--CCTTGGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCCGG RH14 Gladiolus CGAAATCGGTAGACGCTACGGACTTGATTGAATTGA-----G--CCTTGGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG RH20 Gladiolus CGAAATCGGTAGACGCTACGGACTTGATTGAATTGA-----G--CCTTGGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCCGG RH49 Gladiolus CGAAATCGGTAGACGCTACGGACTTGATTGAATTGA-----G--CCTTGGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG RH54 Gladiolus CGAAATCGGTAGACGCTACGGACTTGATTGAATTGA-----G--CCTTGGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Anomatheca ????TTCG-TAGACG--ACGGAATTGATTGAATTGA-----G-CCC-TAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Aristea ???????????????????????????????????????????????????????????????????????????????????????????????????? Babiana ??????????????????????CTTTTATG-ATTGA-----GCGCC----TATGGAAA-CTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAA-CCTGG Chasmanthe ???????????????????????TTG-ATG-ATTGA-----G---CTTAGTATGGAAA-CTGCTAAGTGGTAA---CTTTCAAATTCAGAGAAACCCTGG Crocus ????????????????????????TTGATG-ATTGA------CTTAGTATGGAA-CCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Freesia ??????????????????????CTTG-ATG-ATTGA-----G---CTTAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Geissorhiza ???????????????????????TTG-ATG-ATTGA-----G---CTTAGTAAGGAA-CCT-CTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Geosiris ??????????????????????????????TATTGAT----GCCCCTTAGTATGGAA-CCTACTAAGTGTTAA---CTTCCAAATTCAGAGAAACCCTGG Gladiolus ?????????????AATTTGGATCTTGA-TG-ATTGA-----G---CTTGGTATGGAAA-CTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAA-CCTGG Hesperantha ??????????????????????????????????????????????????TATG-A--CGCTTTAACTGCTATGGACTTC--AATTCAGAGAA--CCTGG Ixia ??AAGGGA?T?GT?TTTCCATCTTTG-ATG-ATTGA------CCTT--TATGGAAA-CTGCTAAGTGATAA---CTTCCAAATTCAGAGAAA-CCTGG Klattia ?????????????????????????????G-ATTGA-----GG?CCTT--TATGGAAA-CTGCTAAGTGGTAA---CTTCCAAATTCAGATAA-CCCTGG Lapeirousia ?G-AATCGGTAGACG-TACGGAATTGAT-GAATTG------G--CCTTAGTATGGAA--CTGCTAAGTAGTA----CTTCC-AATTCAGAGAAACCCTGG Micranthus ?G-AATC-GTAGACGCTACGGAATTGAT-GAATTGA------CCTTTGTATGGAAACCTGCTAAGTGGTTA---CTTCCAAATTCAGAGAAACCCTGG Nivenia ???????????????????????????????????????????????????????????????????????????????????????????????????? Patersonia ???????????????????????????????????????????????????????????????????????????????????????????????????? Pillansia ???AATC-GT-GACGCTACGGAATTGATTGAATTGA-----G---CTTAGTATGGAAA-CTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAA-CCTGG Radinosiphon ????????????????TTTGACTTGCCTTTAAATGA-----G--CCTTAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Romulea ?????????????????????GACTTGATGAATTGA-----G--CCTTAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Savannosiphon ?G--ATC-GTAGACG-TACGGACTTGATTG-ATTGA-----G--CCTTAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Schizostylis ???AATCG-T-GA-GCTACGGACTTGATTGAATTGA-----G--CCTTAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Sparaxis ???????????????????TTGCTTGTTTTATATGA-----G--CCTTAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Syringodea ????????GGGGAA-CT-----CTTGATT-AATTCA-----G---CTTAGTATGGAAA-CTACTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Thereianthus ???????GGTAGACGGTACGGACTTGATTG-ATTGATTCAT----CTTAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Tritonia ??TTTGATGATTGACCTTTTATTGAGA-TG-ATTGA------CCTTT-TATGGAAA-CTGCTAAGTGGTA----CTTCCAAATTCAGAGAA--CCTGG Tritoniopsis ?????????TTCGTAGAGTACGGATTGATGAATTGA-----G---CTT?GTATGGAAA-CTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAA-CCTGG Watsonia ????ATC-GTAGACGCACGGAATTTGATTGAAT-G------GCCCCTTAGTATGGAAACCTGCTAAGTGGTAA---CTTCCAAATTCAGAGAAACCCTGG Witsenia ????????????????????????????????????????????????????????????????????????????????????????????????????

46 Appendices G. illyricus in Britain 110 120 130 140 150 160 170 180 190 200

New Forest GladiolusAAAAAAAAA-GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT RH14 Gladiolus AAAAAAAAAAGGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT RH20 Gladiolus AAAAAAAAA-GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT RH49 Gladiolus AAAAAAAAA-GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT RH54 Gladiolus AAAAAAAAAAGGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT Anomatheca AAAAAAAA--GGGGCAATCC-TGAGCCAAATTTTTATTTTTGAGAAAACAA------AAACTAGAATAAAAAAAAGGATAGGTGCAGAGACTCAAT Aristea ???????????????????????????????????????????????????????????????????????????????????????????????????? Babiana AAAAAA----GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Chasmanthe AAAAAAAAG-GGGGCAATCC-TGAGCCAAATCTTGATTTG-GAGAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Crocus AAAAAAAA-GGGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Freesia AAAAAAAA--GGGGCAATCC-TGAGCCAAATCTTTATTTTTGAGAAAACAA------AAACTAGAAAAAAAA---GGATAGGTGCAGAGACTCAAT Geissorhiza AAAAAAAAGGAGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAA--A------AAACGAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Geosiris AAAAAAAG--GGGGCAATCC-TGAGCCAAGTCTTTATTTTTAACAAA-CAA-GGTTTTTTAAAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT Gladiolus AAAAAAAAAGGGGGCAATCC-TGAGCCAAATCTTTATTTG-GAGAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Hesperantha AAAAA------GGGCAATCC-TGAGCCAAATCTTTATTT--GATAAAACAA------AAAACTAGAATAAAAAA---GATAG-TGCAGAGACTCAAT Ixia AAAAAAAAG-GGGGCGATCC-TGAGCCAAATCTTTATTTT-GAGAAAA-AA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT Klattia AAAAAAGG--GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAAAGGTTTT-AAAAACTACAATAAAAAAG-GGATAGGTGCAGAGACTCAAT Lapeirousia AAAAAAA--AGGGGCCATCCCTGAGCCAAATCTTTATTTT-GATAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Micranthus AAAAAAAAG-GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAA------AAACTAGAATAAAAAAA--GATAGGTGCAGAGACTCGAT Nivenia ???????????????????????????????????????????????????????????????????????????????????????????????????? Patersonia ???????????????????????????????????????????????????????????????????????????????????????????????????? Pillansia AAAAAAAAG-GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Radinosiphon AAAAAAAA-AGGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Romulea AAAAAAAAA-GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Savannosiphon AAAAAAAGG-GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Schizostylis AAAAAAGAAAGGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT Sparaxis AAAAAAAAAGGGGGCAATCC-TGAGCCAAATCTTTGTTTT-AAGAAAACAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT Syringodea AAAAAAAAA-GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Thereianthus AAAAAAAA-GGGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT Tritonia AAAAAAAA-GGGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAACAA------AAACTAGAATAAAAAA--GGATAGGTGCAGAGACTCAAT Tritoniopsis AAAAAAAGG-GGGGCAATCC-TGAGCCAAATCTTTATTTTT-AGAAAACAA------AAACTAGAATAAAAATA-GG-----TGCAGAGACTCGAT Watsonia AAAAAAAG--GGGGCAATCC-TGAGCCAAATCTTTATTTT-GAGAAAA------AAACGAGAATAAAAAAA-GGATAGGTGCAGAGACTCAAT Witsenia ???????????????????????????????????????????????????????????????????????????????????????????????????? 210 220 230 240 250 260 270 280 290 300

New Forest GladiolusGGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------RH14 Gladiolus GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------RH20 Gladiolus GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------RH49 Gladiolus GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------RH54 Gladiolus GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Anomatheca GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Aristea ???????????????????????????????????????????????????????????????????????????????????????????????????? Babiana GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Chasmanthe GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTATTGGTAGCCGAAATCTTTCTATCGAAATGAAA------Crocus GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Freesia GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Geissorhiza GGAAGATGTTCTAACGAATGG------AGTTGACTACGTTGTGTTAGTAGCCGGAATCTTTCTATCGAAATGAAA------Geosiris GGAAGCTGTTCTAACGAATGG------GGTTGACTACGTTGTGTTGGTAGCCGGAACCCTTCTATCGAAATTAAA------Gladiolus GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Hesperantha GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Ixia GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Klattia GGAAGCTGTTCTAACGAATGTAATGGAAACTGTTCTAACGAATGTAGTTGACTACGTTGTGTTGGTAGCCGGAATCCTTCTATCGAAATTAAAAAAAAAA Lapeirousia GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Micranthus GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGTCGGAATCTTTCTATCGAAATGGAA------Nivenia ???????????????????????????????????????????????????????????????????????????????????????????????????? Patersonia ???????????????????????????????????????????????????????????????????????????????????????????????????? Pillansia GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Radinosiphon GGAAGTTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Romulea GGAAGCTGTTCTAACGAATGG------AGTTGACTACCTTGTGTTGGTAACCGGAATCTTTCTATCGAAATGAAA------Savannosiphon GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Schizostylis GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Sparaxis GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Syringodea GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATAAAA------Thereianthus GGAAGCTGTTCTAACGAATGG------GGTTGACTACGTTGTGTTGGTAGCCGGAATTTATCTATCGAAATGAAA------Tritonia GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Tritoniopsis GGA?GCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Watsonia GGAAGCTGTTCTAACGAATGG------AGTTGACTACGTTGTGTTGGTAGCCGGAATCTTTCTATCGAAATGAAA------Witsenia ????????????????????????????????????????????????????????????????????????????????????????????????????

47 Appendices G. illyricus in Britain 310 320 330 340 350 360 370 380 390 400

New Forest Gladiolus------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA RH14 Gladiolus ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA RH20 Gladiolus ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA RH49 Gladiolus ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA RH54 Gladiolus ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Anomatheca ------GATAGATAAGGAT------ATACCGAATTCGTATACATATT-----ACCATATCAAAATCAAACGATTA Aristea ???????????????????????????????????????????????????????????????????????????????????????????????????? Babiana ------GATA----AGGAT------ATACCTAATTCGTATACATATTT----ACCATATCA------AACGATTA Chasmanthe ------GATA----AGGAT------ATACCTAATTCGTATACATA------TCA------AACGATTA Crocus ------GATA----AGGAT------ACACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Freesia ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Geissorhiza ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Geosiris ------GA-A----AGGATAACACTAACACTATTAT--CTAATACGTATACATATTAACTAACC-TATCA------AACGATTA Gladiolus ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Hesperantha ------GATA----AGGAT------ATACCGAATTCGTATACATATT-----ACCATATCA------AACGATTA Ixia ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Klattia AAAACTATCGAAATGAAAGAAAGA-A----AGGATAAC--T----CTAT-ATACCTAATACGTATACATATT-----ACCATATCA------AACGATTA Lapeirousia ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA---T--AACGATTA Micranthus ------GATA----AGGAT------ATACCTAATTCGTATATATATT-----ACCATATCA---T--AACGATTA Nivenia ???????????????????????????????????????????????????????????????????????????????????????????????????? Patersonia ???????????????????????????????????????????????????????????????????????????????????????????????????? Pillansia ------GATA----AGGAT------ATACCTAATTCGTAGACATATT-----ACCATATCA---T--AACGATTA Radinosiphon ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Romulea ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Savannosiphon ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----TCCATATCA---T--AACGATTA Schizostylis ------GATA----AGGAT------ATACCGAATTCGTATACATATT-----ACCATATCA------AACGATTA Sparaxis ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AATGATTA Syringodea ------GATA----AGGAT------ATACCGAATTCGTATACATATT-----ACCATATCA------AACGATTA Thereianthus ------GATA----AGGAT------ATACCTAATTCGTATATATATT-----ACCATATCA---T--AACGATTA Tritonia ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA------AATGATTA Tritoniopsis ------GATA----AG-AT------ATACCTAATTCGTATACATATT-----ACCATATCA------AACGATTA Watsonia ------GATA----AGGAT------ATACCTAATTCGTATACATATT-----ACCATATCA---T--AACGATTA Witsenia ???????????????????????????????????????????????????????????????????????????????????????????????????? 410 420 430 440 450 460 470 480 490 500

New Forest GladiolusATCATGACCCAAATC-ATTATAT-ATATAT------GCAA---AATGGGG---AG--AAATTTTGGATCTATTCCAAT-C------GA--GAAGAATCGA RH14 Gladiolus ATCATGACCCAAATC-ATTATAT-ATATAT------GCAA---AATGGGG---AG--AAATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA RH20 Gladiolus ATCATGACCCAAATC-ATTATAT-ATAT------GCAA---AATGGGG---AG--AAATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA RH49 Gladiolus ATCATGACCCAAATC-ATTATAT-ATATAT------GCAA---AATGGGG---AG--AAATTGTGGATCTATTCCAAT-C------GA--GAAGAATCTA RH54 Gladiolus ATCATGACCCAAATC-ATTATAT-ATATAT------GCAA---AATGGGG---AG--AAATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Anomatheca ATCATGACCCAAATCCATTATAT-ATATATATAT--GCAA---AATGGGG---AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Aristea ???????????????????????????????????????????????????????????????????????????????????????????????????? Babiana ATCATGACCCAAATCCATTATAT-ATATAT------GCAA---AATGGGGGGGAG------TCTAT-CCAAT-C------GA--GAAGAATCGA Chasmanthe ATCATGACTCAAATCCATTATAT-ATATATAT----GCAA---AATGGGG---AG------ATCTATTCCAAT-C------GA--GACGAATCGA Crocus ATCATGACCCAAATCCATTATAT-ATATATAT----GCAA---AA-GGGGG--AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Freesia ATCATGACCCAAATCCATTATAT-ATATAT------GCAA---AATGGGG---AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Geissorhiza ATCATAACCCAAATCCATTATATAATATATATATT-GCAACAAAATGGGGG--AG------ATCCATTCCAAT-C------GA--GAAGAATCAA Geosiris ATCACGATCCAAATCCATTATAT-ATAT------GCAA---AATGC-----AGAGTTTTTGTGGATCTATTCCAAT-CTAAATTGAAGGAAGAGTCGA Gladiolus ATCATGACCCAAATC-ATTATAT-ATATAT------GCAA---AATGGGG---AG--AAATTGTGGATCTATTC------GA--GAAGAATCGA Hesperantha ATCATGGCCCAAATCCATTATAT-ATATATATAT--GCAACAAAATGGGGG--AG------ATCCATTCCAAT-C------GA--GAAGAATCGA Ixia ATCATGACCCAAATCCATTATAT-ATATATAT----GCAA---AATGGGGG--AG------ATCTATTCCAAT-C------GA--GAAGAATCGA Klattia ATCATGACCCAAATCCAT-ATAT-ATAT------GCAA---AATGG-----AGAGCTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCCA Lapeirousia ATCATGACCCAAATCCATTATAT-ATATAT------GCAA---AATGGGG---AG--TTATTGTGGATCTATTCCAAT-C------GA--GACGAATCGA Micranthus ATCATGACCCAAA------ATAT-ATATATATATATGCAA---AATAGGG---AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Nivenia ???????????????????????????????????????????????????????????????????????????????????????????????????? Patersonia ???????????????????????????????????????????????????????????????????????????????????????????????????? Pillansia ATCATGACCCAAATCCATTATAT-ATATAT------GCAA---AATGGG----AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Radinosiphon ATCATGACCCAAATCCATTATAT-ATATAT------GCAA---AATGGGGG--AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Romulea ATTATGACCCAAATCCATTATAT-ATATAT------GCAA---AATGGGGG--AG--TTATTGTGGATCTATTCCAATTCC-AATTGA--GGAGAATCGA Savannosiphon ATCATGACCCAAATCCATTATAT-ATATAT------GCAA---AATGGGG---AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Schizostylis ATCATGACCCAAATCCATTATAT-ATATATA------CAACAAAATGGGGG--AG------ATCCATTCCAAT-C------GA--GAAGAATCGA Sparaxis ATCATGACCCAAATCCATTATAT-ATATATATATATGCAA---AATGGGGT--AG------ATCTATTCCAAT-C------GA--GAAGACTCGA Syringodea ATCATGACCCAAATCCATTATAT-ATATAT------GCAA---AA-GGG----AGAGTTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Thereianthus ATCATGACCCAAATCCATTATAT-ATATAT------GCAA---AATAGGG---AG--TTATTGTGGATCTATTCTAAT-C------GA--GAAGAATCGA Tritonia ATCATGACCCAAATCCATTATAT-ATAT------GCAA---AATGGGGG--AG------ATCTATTCCAAT-C------GA--GAAGAATCGA Tritoniopsis ATCATGACCCAAATCCATTATAT-ATATATATAT--GCAA---AATGGGG---AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Watsonia ATCATGACCCAAATCCATTATAT-ATATAT------GAAA---AATGGGG---AG--TTATTGTGGATCTATTCCAAT-C------GA--GAAGAATCGA Witsenia ????????????????????????????????????????????????????????????????????????????????????????????????????

48 Appendices G. illyricus in Britain 510 520 530 540 550 560 570 580 590 600

New Forest GladiolusATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG RH14 Gladiolus ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG RH20 Gladiolus ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG RH49 Gladiolus ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG RH54 Gladiolus ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Anomatheca ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAACGGATTAATCGTTAATTAATCGTTAAGAGAATAAAGAGAGAG Aristea ???????????????????????????????????????????????????????????????????????????????????????????????????? Babiana ATATTCGGTGATCAAATCTAT-CTATACATTCCAGGGTTTGATAGATCTTTTGAAAAACAGATTAATCG------GA--AGAGAATAAAGAGAGAG Chasmanthe ATATTCGGTGATCAAATCTATTCTAT-CATTCCAGGGTTTGATAGATCTTTTGAAAAACGGATTAATCG------AA--AGAGAATAAAGAGAGAG Crocus ATATTCGGCGATCAAATCTATTCTAT-CATTCCAGAGTTTGATAGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Freesia ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Geissorhiza ATATTCGGTGATCGAATCTATTCTAT-CATTCCAGAGTTTTATAGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Geosiris ATATTCAGTGATCAAAT-TATT-----CATTCCAGAGTTTGATAGATCTTTTGAAAAACGGATTAATTG------GA--AGAGAATAAAGAGAGAG Gladiolus ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATGGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Hesperantha ATATTCGGTGATCAAA-CTATTCTAT-CATTCCAGAGTTTTATAGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Ixia ATATTTGGTGATCAAATCTATTCTAT-CATTCCAGGGTTTGATAGATCTTTTGAAAAACAGATTAATCG------GA--AGAGAATAAAGAGAGAG Klattia ATATTTGGTGATCAATT-TAT------CATTCCAGAGTTTGATAGATCTTT-GACACACGGATTAATCG------GAT-AGAGAATAAAGAGAGA- Lapeirousia ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAATGGATTAATCG------GA--AGAGAATAAAGAGAGAG Micranthus ATATTCGGTGATCAAATCCAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAGCAGATTAATCG------GA--AGAGAATAAAGAGAGAG Nivenia ???????????????????????????????????????????????????????????????????????????????????????????????????? Patersonia ???????????????????????????????????????????????????????????????????????????????????????????????????? Pillansia ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAACAGATTAATCG------GA--AGAGAATAAAGAGAGAG Radinosiphon ATATTCGGTGATCAAATCTATTCTAT-CATTCCAGAGTTTGATAGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Romulea ATATTCGGTGATCAAATCTATTCTAT-CATTCCGGAGTTTGATAGATCTTTTGAAAAACAAATTAATCG------GA--AGAGAATAGAGAGAGAG Savannosiphon ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAATGGATTAATCG------TA--AGAGAATAAAGAGAGAG Schizostylis ATATTCGGTGATCAAACCTATTCTAT-CATTCCAGAGTTTTATAGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Sparaxis ATATTCGGTGATCAAATCTATTCTAT-CATTCCAGGGTTTGATAGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Syringodea ATATTCGGCGATCAAATCTATTCTAT-CATTCCAGAGTT?GATAGATCTTTTGAAAAACGGATTAATCG------GA--AGAGAATAAAGAGAGAG Thereianthus ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAACAGATTAATTG------GA--AGAGAATAAAGAGAGAG Tritonia ATATTCGGTGATCAAATCTATTCTAT-CATTCCAGGGTTTGATAGATCTTTTGAAAAACAGATTAATCG------GA--AGAGAATAAAGAGAGAG Tritoniopsis ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAATGGATTAATCG------GA--AGAGAATAAAGAGAGAG Watsonia ATATTCGGTGATCAAATCTAT------CATTCCAGAGTTTGATAGATCTTTTGAAAAACAGATTAATCG------GA--AGAGAATAAAGAGAGAG Witsenia ???????????????????????????????????????????????????????????????????????????????????????????????????? 610 620 630 640 650 660 670 680 690 700

New Forest GladiolusTCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA RH14 Gladiolus TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA RH20 Gladiolus TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA RH49 Gladiolus TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA RH54 Gladiolus TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Anomatheca TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Aristea ??????????????????????????????????????????????????????????????????????????????GGTTCAAGTCCCTCTATCCCCA Babiana TCCCATTATACATGTCAATAACGACAACAATGAAATTTATAGTAAGAAGGGAAATCCGTTGAATTTAGAAATTGTGAGGGTTCAAGTCCCTTTATCCCCA Chasmanthe TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCG?CGACTTTAAAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Crocus TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Freesia TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Geissorhiza TCCCATTTTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Geosiris TCCCATTCTACATGTCAATAACGACAACAATAAAATTGATAGTAA-AAGGAAAATCCGTCGACTTTCGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Gladiolus TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Hesperantha TCCCATTTTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGGAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Ixia TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Klattia TCCCAT-CTACATGTTA-TAACGACAACA-TGAA-TT-ATAGTAA--AG---AATCG-TCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Lapeirousia TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Micranthus TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Nivenia ??????????????????????????????????????????????????????????????????????????????GGTTCAAGTCCCTCTATCCCCA Patersonia ???????????????????????????????????????????????????????????????????????????????GTTCAAGTCCCTCTATCCCCA Pillansia TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Radinosiphon TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Romulea TCCCATTCTACATGTCAATAACGACCACAATGGAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Savannosiphon TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Schizostylis TCCCGTTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Sparaxis TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Syringodea TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCG?GAGGGTTCAAGTCCCTCTATCCCCA Thereianthus TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Tritonia TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGT-CAAGTCCCTCTATCCCCA Tritoniopsis TCCCATTCTACATGTAAATAACGACAATGATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Watsonia TCCCATTCTACATGTCAATAACGACAACAATGAAATTTATAGTAA-AAGGAAAATCCGTCGACTTTAGAAATCGTGAGGGTTCAAGTCCCTCTATCCCCA Witsenia ??????????????????????????????????????????????????????????????????????????????GGTTCAAGTCCCTCTATCCCCA

49 Appendices G. illyricus in Britain 710 720 730 740 750 760 770 780 790 800

New Forest GladiolusATAA-AAAGTCCATTTTACTTCC-TAACAATTTCTA------TTATTCTATTCTTTTT------RH14 Gladiolus ATAA-AAAGTCCATTTTACTTCC-TAACAATTTCTA------TTATTCTATTCTTTTT------RH20 Gladiolus ATAA-AAAGTCCATTTTACTTCC-TAACAATTTCTA------TTATTCTATTCTTTTT------RH49 Gladiolus ATAA-AAAGTCCATTTTACTTCC-TAACAATTTCTA------TTATTCTATTCTTTTT------RH54 Gladiolus ATAA-AAAGTCCATTTTACTTCC-TAACAATTTCTA------TTATTCTATTCTTTTT------Anomatheca ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Aristea ACAA-AAAGCCCATTTTACTTCC-TAACTATTGCTA------TTATTATATTCTTTTTTTT-----CATCTTCCTCCATCAACATCAGCGA Babiana ATAA-AAAGTACATTTTAATTCCCTAACGATTTCTA------TTATTTTATATATTTTTTTTTTT------Chasmanthe ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTTTTTTT------Crocus ATAATAAAGTCCATTTTACTTTC-TAACGATTTCTA------TTATTCTATTCTTTTT------Freesia ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Geissorhiza ATAA-AAAGTCCATTTAACTTCC-TCACGATTTCTA------TTATTCTATTTTTTTTTTTT------Geosiris ATAA-AAAGCCTCTTTTACTTCC-TAACTATTTCTA------TTATTATATTATTTTTT------CATCTTCATCCATCAGCATCCGCGG Gladiolus ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Hesperantha ATAA-AAAGCCCATTTAACTTCC-TCACGATTTCTA------TATTCTATTTTTTT------Ixia ATAA-AAAGTCCATTTGACTTCC-TAACTATTTCTATCTAACTATTTCTAATTATTCTATTTTTTTT------Klattia ATAA-AAAGTCCATTTTACTTCCC-AACTATTTCTA------TTATATTCTTGTTTTTTTT--CATCTTCCTCGATCAGCATCAGTGG Lapeirousia ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Micranthus ATAA-AAAGCCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Nivenia ATAA-AAAGTCCATTTTACTTCC-TCACGATTTCTATTA------TTATTATATTCTTTTTTTTT----CATCTTCCTCGATCAGCATCAGTGG Patersonia A-AA-AAAGCCCATTTTACTTCC-TAACTATTT-AATTAAA------TTTTTATATTCTTCTTTTTT----CATCTTCATCCAT------Pillansia ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Radinosiphon ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Romulea ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Savannosiphon CTAA-AAAGTCCATTTTACTTCC-TAACGATTTCTC------TTATTCTATTCTTTTT------Schizostylis ATAA-AAAGTCCATTTTACTTCC-TCACGATTTCTA------TTATTCTAATTTTTTT------Sparaxis AGAA-AAAGTCCATTTTACTTAC-TAACGATTTCTA------TTATTCTATTTTTTTT------Syringodea AAAA-AAAGTCCATTTGACTTCC-TAATGATTTCTA------TTATTCTATTCTTTATT------Thereianthus ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------CTATTCTATTCTTTTT------Tritonia ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTTTTTTTTT------Tritoniopsis ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCTA------TTATTCTATTCTTTTT------Watsonia ATAA-AAAGTCCATTTTACTTCC-TAACGATTTCAT------ATATTCTATTCTTTTT------Witsenia ATAA-AAAGTCCATTTTACTTCC-TAACTATA-CTATTT------CTATTATATTCTTTTTTTTTTTTTCATCTTCCTCGATCAGCATCAGTGG 810 820 830 840 850 860 870 880 890 900

New Forest Gladiolus------RH14 Gladiolus ------RH20 Gladiolus ------RH49 Gladiolus ------RH54 Gladiolus ------Anomatheca ------Aristea TTCCAACAAAATGAAATATCTTTATCATTAATTCTAT--TCTTTCACAAACAGATCCGAACAGAAGTCTTTGGATCTTATCCCAATTAGGTTTGGATAGA Babiana ------Chasmanthe ------Crocus ------Freesia ------Geissorhiza ------Geosiris TTCCAACAAAATGACATATCTTTCTCATTAATTCTATACTCTTTCACAAATAGACCCGAAAAGAAATCTTTGGATCTTATCCCAATTGGGTTTGGATAGA Gladiolus ------Hesperantha ------Ixia ------Klattia CTCCAACAAAATTCAATATCTTTCTCATTAATTCTA--CTCTTTCGCAAACAGATCCGAACAGAAATCTTTGGATCTTATCCCAATTGGGTTTGGATAGA Lapeirousia ------Micranthus ------Nivenia TTCCAACAAAATTCAATATCTTTCTCATTAATTCTA--CTCTTTCGCAAACAGATCCGAACAGAAATCTTTGGATCTTATCCCAATTGGGTTTGGATAGA Patersonia ------AATCTTTAGATCTTATCCCAATTTGGTTTGGATAGA Pillansia ------Radinosiphon ------Romulea ------Savannosiphon ------Schizostylis ------Sparaxis ------Syringodea ------Thereianthus ------Tritonia ------Tritoniopsis ------Watsonia ------Witsenia TTCCAACAAAATTCAATATCTTTCTCATTAATTCTA--CTCTTTCGCAAACAGATCCGAACAGAAATCTTTGGATCTTATCCCAATTGGGTTTGGATAGA

50 Appendices G. illyricus in Britain 910 920 930 940 950 960 970 980 990 1000

New Forest Gladiolus------RH14 Gladiolus ------RH20 Gladiolus ------RH49 Gladiolus ------RH54 Gladiolus ------Anomatheca ------Aristea TATGATACTTGTACAAATGAGCATATATGGGAAAGGGATTCTCATTA------Babiana ------Chasmanthe ------Crocus ------Freesia ------Geissorhiza ------Geosiris TATGATACCTGTACAAATGAACATAT------GGAATTTTCATTATTGAA------TCATTCACAGTCCATATCATTATCCTTACGTTTACAAA Gladiolus ------Hesperantha ------Ixia ------Klattia TATGATACCTGTACAAATGAGCATATATGGGAAAGGAATTCTCATTATTGAA------TCATTCACAGCCCATATCATTATCCTTACATTTCCAAA Lapeirousia ------Micranthus ------Nivenia TATGATACCTGTACAAATGAGCATATATGGGAAAGGAATTCTCATTATTGAA------TCATTCACAGTCCATATCATTATTCTTACGTTTCCAAA Patersonia TATGATACCTATACAAATAAGCATATATGGGCAAGGAATTCCCATTATTGAATTAATAATAATAATTCACAGTCCATATCATTATCCTTACATTTACAAG Pillansia ------Radinosiphon ------Romulea ------Savannosiphon ------Schizostylis ------Sparaxis ------Syringodea ------Thereianthus ------Tritonia ------Tritoniopsis ------Watsonia ------Witsenia TATGATACCTGTACAAATGAGCATATATGGGAAAGGAATTCTCATTATTGAA------TCATTCACAGCCCATATCATTATCCTTACGTTTCCAAA 1010 1020 1030 1040 1050 1060 1070 1080 1090 1100

New Forest Gladiolus------GAATGAAGATCTAAAACTCAGAAATTTT------GGGGACTGTGCC-----AAA------TTTTTTAATACTTT----- RH14 Gladiolus ------GAATGAAGATCTAAAACTCAGAAATTTT------GGGGACTGTGCC-----AAA------TTTTTTAATACTTT----- RH20 Gladiolus ------GAATGAAGATCTAAAACTCAGAAATTTT------GGGGACTGTGCC-----AAA------TTTTTTAATACTTT----- RH49 Gladiolus ------GAATGAAGATCTAAAACTCAGAAATTTT------GGGGACTGTGCC-----AAA------TTTTTTAATACTTT----- RH54 Gladiolus ------GAATGAAGATCTAAAACTCAGAAATTTT------GGGGACTGTGCC-----AAA------TTTTTTAATACTTT----- Anomatheca ------GAATTAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----GAA------TTTTTGAATACTTT----- Aristea ------ATGAAGATCTAAAACTAAGAAATTTC------AGGGACTATGTC-----AAA------TTTTTGAATACTTT----- Babiana ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAAGCCAAATTTTTGAATACTTT----- Chasmanthe ------GAATGAAGATCTAAAACTAAGGAATTTC------GGGGACTATGCC-----AAAGCCAAATTTTTGAATACTTT----- Crocus ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Freesia ------GAATTAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----GAA------TTTTTGAATACTTT----- Geissorhiza ------ATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Geosiris TACAAAGAAAGTATTCTTTTTGAATGAAGATCTAAAACTAAGAAATTTCATGGACATGGACTATGTC-----AAA------TTTTT-AATACTTTGCTCT Gladiolus ------GAATGAAGATCTAAAACTCAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Hesperantha ------GGATGGAGATCTAAAACTAAG-AATTT------GGGGACTATGCC-----AAAGCCAAAATTTTTAATACTTT----- Ixia ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAAGCCAAATTTTTTA----TTT----- Klattia ------GAAAGTCTTCTTTTTGAATAAAGATCTAAAACTAAGAAATTTC------GGGTGCTATACC-----AAA------TTTTTTAATACTTT----- Lapeirousia ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Micranthus ------GAATGAAGATCTAAAACTAAGAAATTTCA-----GGGGACTATGCC----CAAA------TTTTTTAATACTTT----- Nivenia --GAAAGAAAGTCTTCTTTTTGAATGAAGATCTAAAACTAAGAAATTTC------GGGTACTATACCATACCAAA------TTTTTTAATACTTT----- Patersonia ------GAAGGTCCTCTTTTTGAATGAAGATTTAAGACTAAGAAATTTC------GGGGACTATGTC-----AAA------TTTGTTAATACTTT----- Pillansia ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Radinosiphon ------GAAGGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATGCTTT----- Romulea ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCT-----AAA------TTTTTTAATACTTT----- Savannosiphon ------GAATGAGGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Schizostylis ------GAATGAAGATCTAAAACTAAGAAATTTT------TTGGACTATGCC-----AAA------TTTTTTAATACTTT----- Sparaxis ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTGTGCC-----AAAGCCAAATTTTTTAATACTTT----- Syringodea ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAAGCCAAATTTTTTAATACTTT----- Thereianthus ------GAATGAAGATCTAAAACTAAGAAATTTCA-----GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Tritonia ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAAGCCAAATTTTTTAATACTTT----- Tritoniopsis ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Watsonia ------GAATGAAGATCTAAAACTAAGAAATTTC------GGGGACTATGCC-----AAA------TTTTTTAATACTTT----- Witsenia ------GAAAGTCTTCTTTTTGAATAAAGATCTAAAACTAAGAAATTTC------GGGTGCTATACC-----AAA------TTTTTTAATACTTT-----

51 Appendices G. illyricus in Britain 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200

New Forest GladiolusTGTAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTATATAGGATGATGCGCGGTAAATAGTCGGG------ATAG------RH14 Gladiolus TGTAGTCTCTT-----TAATTTCC----ATAG-----AC-CCAA-GTACTCTATATAGGATGATGCGCGGGAAATAGTCGGG------ATAG------RH20 Gladiolus TGTAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTATATAGGATGATGCGCGGGAAATAGTCGGG------ATAG------RH49 Gladiolus TGTAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTATATAGGATGATGCGCGGGAAATAGTCGGG------ATAG------RH54 Gladiolus TGTAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTATATAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Anomatheca GGCAGTCTCTT-----TAATTTCC----ATAG-----AC-CAAA-GTACTCTAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Aristea ------T-----TAATTCCC----ATAG-----AT-ACAA-GTACTCTAC-TAGAATGATGCGCGGGAAATGGTCGGG------ATAG------Babiana GGCAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Chasmanthe GGCAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTAT-TAGGATGATGCGCGGGAAATTGACGGG------ATAG------Crocus GGCAGTCTCTT-----TAATTTCC----ATAG-----AT-TCAA-GTACTCTAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Freesia GGC-----CTT-----TCATTTCCC---ATAG-----AT-CCAA-GTACTCTAT-TAGGATGATGCGCGGG-AATAGTCGGG------ATAG------Geissorhiza GGCAGTCTCTT-----TAATTTCC----ATAG-----AT-TCGA-GTACTCCAT-TAGGATGATGCGCGG--AATAGTCGG------ATAG------Geosiris GGTAGTCTCTT-----TAATTTCC----ATAGTAAAGAT-ACAA-GTACTCCAC-TAGGATGATG------AATGGTCGGG------ATAG------Gladiolus TGTAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTATATAGGATGATGCGCGGGAAATAGTCGGG------?TAG------Hesperantha GGCAGTCTCTT-----TAATTTCC----ATAA-----AT-CCAA-GTACTCCAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Ixia GGTAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCCAT-TAGGATGATGCACGGGAAAT??TCGAG------AT-G------Klattia GGTAGTCTCTT-----TAATTTCC----AAAG-----AT-AGAA-GTACTCTAT-TAGGATGATGCGCGGGAAATGGTCGGG------ATAG------Lapeirousia ?GCAGTCTCT?-----TAAT?T------A-AA-----AT????????????????????????????????????????------???????------Micranthus TGCAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAAAGTACTCTAT-TAGGATGATGCACAGG-AATAGTCGGG------ATAG------Nivenia GGCAGTCTCTT-----TAATTTCT----ATAG-----AT-AGAA-GTACTCTAT-TAGGATGATGCGCGGGAAATGGTCGGG------ATAG------Patersonia GGTAGTCTCTT-----TCATTTCC----ATAG-----AT-ACAA-GTACTCCAC-TAGGATGATGCGCGGTAAATGGTCGGG------ATAG------Pillansia GGCAGTCTCTT-----TAATTTCC----ATAG-----ATCCCAA-GTACTCTAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Radinosiphon GGCAGTCTCTT-----TCATTTCC----ATAG-----ATACCAA-GTACTCTAT-TAGGATGATGCGCGGGAAATAGTCGGG------TAG------Romulea GGCAGTCTATT-----TAATCTCC----ATAG-----AT-CCAA-GTACTCTAT-AAGGATGATGCGCGGGAAGGAGTCGGG------ATAG------Savannosiphon GGCAGTCTCTT-----TAATTTCC----ATAG-----AT-ACAA-GTACTCTAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Schizostylis GGCAGTCTCTT-----TAATTTCC----ATAA-----AT-CCGA-GTACTCCAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Sparaxis GGCAGTCTCTT-----TAATTTCCC---ATAG-----AT-CCAA-GTACTCCAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Syringodea GGCAGTCTCTT-----TAATTTCCA---ATAG-----AT-TCAA-GTCATCCAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Thereianthus GGCAGTCTCTT-----TAATTTCC----ATAG-----ATCCCAA-GTACTCTAT-TAGGATGATGTGCGGGAAATAATCCGG------ATAG------Tritonia GGCAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCCAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Tritoniopsis GGCAGTCTCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTAT-TAGGATGATGCGCGGGAA?TAGTCGCGCGG?TCATAGTA?GGG?G Watsonia GGCAGTATCTT-----TAATTTCC----ATAG-----AT-CCAA-GTACTCTAT-TAGGATGATGCGCGGGAAATAGTCGGG------ATAG------Witsenia GGTAGTCTCTT-----TAATTTCC----AAAG-----AT-AGAA-GTACTCTAT-TAGGATGATGCGCGGGAAA-GGTCGGG------ATAG------1210 1220 1230 1240 1250 1260 1270

New Forest Gladiolus CTC-AGTTGGT-AGAG------CAGAGGACTGAAAA------TCCTCGTGTCACCAGTTCAAAT RH14 Gladiolus CTC-AGTTGGT-AGAG------CAGAGGACTGAAAA------TCCTCGTGTCACCAGTTCAAAT RH20 Gladiolus CTC-AGTTGGT-AGAG------CAGAGGACTGAAAA------TCCTCGTGTCACCAGTTCAAAT RH49 Gladiolus CTC-AGTTGGT-AGAG------CAGAGGACTGAAAA------TCCTCGTGTCACCAGTTCAAAT RH54 Gladiolus CTC-AGTTGGT-AGAG------CAGAGGACTGAAAA------TCCTCGTGTCACCAGTTCAAAT Anomatheca CTC-AGTTGGT-AGAG------CAGAGGACTGAA------TAATCCCTTTTCC---CAAAAT Aristea CTC-AGTT-GT-AGAG------GAG-GGACTGAAAAA??????????????????????????? Babiana CTC-AGTTAAT-AG-G------CAGA-GACTGAAAA------GC????????????????? Chasmanthe CTC-AGTTGGT-AGAG------CAGA-GACTGAA------G-G?????????????? Crocus CTCCAGTTG-T-AGAG------CAGA-GACTGAAAAA------AATT Freesia CTC-AGT--GT-AGAG------CAGA-GACTGAA?????????????????????????????? Geissorhiza CTC-AGTT-GT-AGAG------CAGA-GA-TGA??????????????????????????????? Geosiris CTC-AGTTGGT-AGAG------GAGAGGACTGAA------T------A--A-AT?--AT Gladiolus CTC-AGT?G?--AG?G------CAG?G-?CTGAAAA------AAAT Hesperantha CTC-AGTTGATAAG------CAGAGAACTGAAAA------TATTT Ixia CCC-ATTTAA?-AGGG------CCAGA----TGAAAAAAAT--CCCCTTTCCCCGTTCTATATTT Klattia CTC-AGTTGGT-AGGGAAAGA------CGCAAAGGAAATGAAAA------TCCTCGTTTCACCAGTTCAAAT Lapeirousia ???????????????????????????????????????????????????????????????????????? Micranthus CTC-AATTGGT-A-AG------CA-A-GACTGAAAA------TCCTCGTTTCACCAGTTCAAAT Nivenia CTC-AGTTGGT-AGAG------CGCCGGACTGAAAAA?A????????????????????????? Patersonia CTC-AGTTGGT-AGAG------CAGAGGACTGAAAA------TCCTCGTGTCACCAGTTCAA-T Pillansia CTC-AGTT-GTAAGAG------AGAGGACTGAAAA------TCC--CTGTC-C--GT-CAAAT Radinosiphon CTC-AGTT-AT-A-A------C-GA-GACTGAAAA------AATT Romulea CTC-AGTTAAT-AGAGCCGAGACTGAAAGGCAGA-GACTGAAA------A?ATT Savannosiphon CTC-AGTT-GT-AAACAA------AGA-GACTGAAA------TCCTC-T------TTCAAAT Schizostylis CTC-AGTTG-T-AGAG------CGGAGGACTGAAAAA------TAATT Sparaxis CTC-AGTTGGT-AGAG------CAGA-GACTGAAAA------GCGT------ATTT Syringodea CTC-AGTTGGT-A?AG------CAGA-GACTGAAA------TCCTC-TGTC------AATTT Thereianthus CTC-A-TTG-T-ACAGA------CAGAGGACTGAAAA------TCCTC------AAAT Tritonia CTC-AGTTGGT-AGAG------CAGGCGACTGAAAA------TCC------CC--TT-ATTT Tritoniopsis CTC-A?TT?GT-AAAG------CA?AGGACTGAAAA------TC?-CCTGTC-CCA-TCAAAAT Watsonia CTC?A?T?GG?-A?A------CAGA-GACTGAAAA------TCCTCGTGTCACCA-TTCAAAT Witsenia CTC-AGTT-AA-AGAG------GCCCGGACTGAAAAACAAAA??????????????????????

52 Appendices G. illyricus in Britain

H Unique Haplotype Determination

#!/usr/bin/perl # # ./haplotypes.pl filename start_position end_position # # Takes a NEX* format alignment (generated by MegAlign) and returns a digest # of the haplotypes therein. Start and end positions can be specified so that # the begining and end of the alignment can be ignored. NOTE: gives position # information from the alignment, not each sequence individally! # # examples: # ./haplotypes.pl COX1.NEX # ./haplotypes.pl alignment.NEX 30 723 # # To generate the NEXUS file, open/create an alignment in MegAlign, then # from the menu at the top of the screen, select View->Phylogenic Tree. # Next select File->Save As... then type in a filename as normal, but this # select "Format: PAUP [NEXUS]" using the drop-down menu near the bottom # left. Click OK. Move this file into the same directory if you want, then # use the example command above in a Terminal having "cd"ed to that # directory. To save the output in a file called ’alignment_summary.txt’ # use: # # ./haplotypes.pl alignment.NEX 30 723 > alignment_summary.txt # # and simply miss out the numbers if you want the ends to count. # # *also takes simple fasta-like files and ".aln" alignment files, the latter # being the output of CLUSTAL W (1.83) multiple sequence alignment (align.genome.jp) sub max($$) { ($x,$y)=@_; $x >= $y ? $x : $y; }

$file = $ARGV[0]; print "Working from ’$file’.\n";

# For removing any Mac-style newlines $correct_cmd = "cat $file | perl -pne ’s/ˆM/\\n/g’ > temp; mv temp $file"; $correct_cmd_proper = "cat $file | perl -pne ’s/ /\\n/g’ > temp; mv temp $file"; print "> $correct_cmd\n"; ‘$correct_cmd_proper‘; open(FILE,"<$file") or die "Could not open ’$file’ for reading.\n";

$first_line = 1; $file_type = ’unknown’; $found_char_block = 0; $reading_data = 0; $id = ’id_error’; while () { if ($first_line) { # test file format $first_line = 0; if (/ˆ#NEXUS$/) { $file_type = ’NEXUS’; print "NEXUS file format.\n"; } elsif (/ˆ#NEXUS/) { die("NEXUS file with wrong line termination characters.\nTry > $correct_cmd\n"); } elsif (/ˆCLUSTAL/) { $file_type = ’CLUSTAL’; print "CLUSTAL file format.\n"; } else { $file_type = ’FASTA’; print "Assuming simple fasta-like format.\n"; } }

if ($file_type eq ’NEXUS’) { # prefer not to have many level of indentation if (/BEGIN CHARACTER/) { $found_char_block = 1; } elsif ($found_char_block && /matrix/) { $found_char_block = 0; $reading_data = 1; } elsif ($reading_data && /;/) { $reading_data = 0; }

s/\[[ˆ\]]*\]//g; # remove comments if ($reading_data && !/ˆ\s*$/ && !/matrix/) { /ˆ(\S*)\s*(\S*)/; $id = $1; $seq_frag = $2; $id =˜ s/’//g; # remove quotation marks $data{$id} .= uc($seq_frag); # convert to upper case and append } } elsif ($file_type eq ’CLUSTAL’) { if (/ˆCLUSTAL/ or /ˆ\s/) { # Ignore first line and those starting with space }

53 Appendices G. illyricus in Britain

else { /ˆ(\S*)\s*(\S*)/; $id = $1; $seq_frag = $2; $data{$id} .= uc($seq_frag); # convert to upper case and append } } elsif ($file_type eq ’FASTA’) { chomp; # assume simple fasta-like file if (/ˆ\s*$/) { #print "whitespace---ignoring.\n"; } elsif (/ˆ>/) { s/ˆ>//; $id = $_; #print "Next entry: id = ’$id’\n"; } else { $data{$id} .= uc($_); #print "Data found; adding to ’$id’.\n"; } } else { die "Unknown file type ’$file_type’.\n"; } }

# check lengths are the same... @data_ids = keys %data; $num_sequences = scalar(@data_ids); print "Found $num_sequences sequences.\n";

$highest_count = 0; $mode_length = 0; foreach $i (0..$#data_ids) { $lengths[$i] = length($data{$data_ids[$i]}); $length_counts{$lengths[$i]}++; if ($length_counts{$lengths[$i]}>$highest_count){ $highest_count = $length_counts{$lengths[$i]}; $mode_length = $lengths[$i]; } } if (scalar(keys %length_counts)>1) { print "Mode length = $mode_length\n"; foreach $key (keys %length_counts) { print "$key -> $length_counts{$key}\n"; } } foreach $i (0..$#data_ids) { if ( $lengths[$i]!=$mode_length ) { print "Length ($lengths[$i]) of sequence ’$data_ids[$i]’ is different from the mode: removing from analysis\n"; delete($data{$data_ids[$i]}); } } # recalculate in case of changes @data_ids = keys %data; print "Found an aligned sequence set of $mode_length positions.\n\n";

$start_position = 1; $end_position = $mode_length; if ($#ARGV>0) { $start_position = $ARGV[1]; } if ($#ARGV>1) { $end_position = $ARGV[2]; } print "Scanning sequences from $start_position to $end_position.\n";

# Find variable columns foreach $i ($start_position..$end_position) { foreach $j (1..$#data_ids) { $last_char = substr($data{$data_ids[$j-1]},$i-1,1); $this_char = substr($data{$data_ids[$j]},$i-1,1); if ( $last_char ne $this_char ) { push(@variable_columns, $i); last; } } }

$" = ’, ’; print "Substitutions or indels found in columns:\n @variable_columns\n\n"; print "Context:\n"; $radius = 12; $id = $data_ids[0]; $context = substr($data{$id},max(0,$variable_columns[0]-$radius-1),$radius); $context .= ’*’; $context .= substr($data{$id},$variable_columns[0],$radius); @variable_columns_indices = (0); foreach $i (1..$#variable_columns) { if ($variable_columns[$i]-$variable_columns[$i-1] == 1) { $context = substr($context,0,length($context)-$radius); $context .= ’*’; $context .= substr($data{$id},$variable_columns[$i],$radius);

54 Appendices G. illyricus in Britain

} else { if ($variable_columns_indices[$#variable_columns_indices]==$i-1) { $start_column = ’ ’ x (5+3); } else { $start_column = sprintf("% 5d - ",$variable_columns[$variable_columns_indices[$#variable_columns_indices]]); } printf(" $context \t:$start_column%d\n",$variable_columns[$i-1]); push(@variable_columns_indices,$i); $context = substr($data{$id},max(0,$variable_columns[$i]-$radius-1),$radius); $context .= ’*’; $context .= substr($data{$id},$variable_columns[$i],$radius); } } if ($variable_columns_indices[$#variable_columns_indices]==$#variable_columns) { $start_column = ’ ’ x (5+3); } else { $start_column = sprintf("% 5d - ",$variable_columns[$variable_columns_indices[$#variable_columns_indices]]); } printf(" $context \t:$start_column%d\n",$variable_columns[$#variable_columns]);

# Create mini data matrix foreach $id (keys %data) { foreach $i (@variable_columns) { $var_data{$id} .= substr($data{$id},$i-1,1); } # print "$var_data{$id} <- $id\n"; }

# order @sorted_ids = sort { $var_data{$a} cmp $var_data{$b} } @data_ids;

# pick out unique haplotypes @haplotypes = ( $var_data{$sorted_ids[0]} ); @haplotype_specimens = ( [ $sorted_ids[0] ] ); foreach $i (1..$#data_ids) { if ( $var_data{$sorted_ids[$i]} ne $var_data{$sorted_ids[$i-1]} ) { push( @haplotypes, $var_data{$sorted_ids[$i]} ); push( @haplotype_specimens, [ $sorted_ids[$i] ] ); } else { push( @{$haplotype_specimens[$#haplotype_specimens]}, $sorted_ids[$i] ); } }

# Display results print "Haplotypes:\n"; push(@variable_columns_indices,$#variable_columns+2); $spacer = ’ ’; foreach $i (0..$#haplotypes) { printf("% 3d: ",$i+1); foreach $j (0..($#variable_columns_indices-1)) { $number = $variable_columns_indices[$j+1]-$variable_columns_indices[$j]; print $spacer; print substr($haplotypes[$i],$variable_columns_indices[$j],$number); } print "\n"; } print "\n"; foreach $i (0..$#haplotype_specimens) { print ($i+1)."."; @sorted_names = sort { $a cmp $b } @{$haplotype_specimens[$i]}; foreach $j (0..scalar(@sorted_names)) { print "\t$sorted_names[$j]\n"; } } print "Distance matrix:\n"; $a = 5; print ’ ’ x ($a+1); foreach $i (1..$#haplotypes+1) { printf(’% 3d ’,$i); } print "\n\n"; foreach $i (0..$#haplotypes) { printf("% ${a}d ",$i+1); print ’ ’ x $i; print ’ - ’; foreach $j ($i+1..$#haplotypes){ $count = 0; foreach $k (0..length($haplotypes[$j])) { $count++ if substr($haplotypes[$j],$k,1) ne substr($haplotypes[$i],$k,1); } printf(’% 3d ’,$count); } print "\n"; }

55 References G. illyricus in Britain

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